Methods and reagents for the inhibition of hepatitis B virus replication

ABSTRACT

The present invention relates to nucleic acid molecules that modulate Hepatitis B virus (HBV) gene expression and HBV replication, and methods thereof. Specifically, the present invention relates to nucleic acid decoy molecules and aptamers that bind to HBV reverse transcriptase and/or HBV reverse transcriptase primer sequences and methods for their use alone or in combination with other therapies. The present invention also relates to nucleic acid molecules that specifically bind the Enhancer I region of HBV DNA and methods for their use.

BACKGROUND OF THE INVENTION

[0001] The present invention concerns compounds, compositions, andmethods for the study, diagnosis, and treatment of degenerative anddisease states related to hepatitis B virus (HBV) infection, replicationand gene expression. Specifically, the invention relates to nucleic acidmolecules used to modulate expression of HBV.

[0002] The following is a discussion of studies relating to hepatitis Bvirus (HBV). The discussion is not meant to be complete and is providedonly for understanding of the invention that follows. The summary is notan admission that any of the work described below is prior art to theclaimed invention.

[0003] Chronic hepatitis B is caused by an enveloped virus, commonlyknown as the hepatitis B virus or HBV. HBV is transmitted via infectedblood or other body fluids, especially saliva and semen, duringdelivery, sexual activity, or sharing of needles contaminated byinfected blood. Individuals may be “carriers” and transmit the infectionto others without ever having experienced symptoms of the disease.Persons at highest risk are those with multiple sex partners, those witha history of sexually transmitted diseases, parenteral drug users,infants born to infected mothers, “close” contacts or sexual partners ofinfected persons, and healthcare personnel or other service employeeswho have contact with blood. Transmission is also possible viatattooing, ear or body piercing, and acupuncture; the virus is alsostable on razors, toothbrushes, baby bottles, eating utensils, and somehospital equipment such as respirators, scopes and instruments. There isno evidence that HBsAg positive food handlers pose a health risk in anoccupational setting, nor should they be excluded from work. Hepatitis Bhas never been documented as being a food-borne disease. The averageincubation period is 60 to 90 days, with a range of 45 to 180; thenumber of days appears to be related to the amount of virus to which theperson was exposed. However, determining the length of incubation isdifficult, since onset of symptoms is insidious. Approximately 50% ofpatients develop symptoms of acute hepatitis that last from 1 to 4weeks. Two percent or less of these individuals develop fulminanthepatitis resulting in liver failure and death.

[0004] The determinants of severity include: (1) The size of the dose towhich the person was exposed; (2) the person's age with younger patientsexperiencing a milder form of the disease; (3) the status of the immunesystem with those who are immunosuppressed experiencing milder cases;and (4) the presence or absence of co-infection with the Delta virus(hepatitis D), with more severe cases resulting from co-infection. Insymptomatic cases, clinical signs include loss of appetite, nausea,vomiting, abdominal pain in the right upper quadrant, arthralgia, andtiredness/loss of energy. Jaundice is not experienced in all cases,however, jaundice is more likely to occur if the infection is due totransfusion or percutaneous serum transfer, and it is accompanied bymild pruritus in some patients. Bilirubin elevations are demonstrated indark urine and clay-colored stools, and liver enlargement may occuraccompanied by right upper-quadrant pain. The acute phase of the diseasemay be accompanied by severe depression, meningitis, Guillain-Barresyndrome, myelitis, encephalitis, agranulocytosis, and/orthrombocytopenia.

[0005] Hepatitis B is generally self-limiting and will resolve inapproximately 6 months. Asymptomatic cases can be detected by serologictesting, since the presence of the virus leads to production of largeamounts of HBsAg in the blood. This antigen is the first and most usefuldiagnostic marker for active infections. However, if HBsAg remainspositive for 20 weeks or longer, the person is likely to remain positiveindefinitely and is now a carrier. While only 10% of persons over age 6who contract HBV become carriers, 90% of infants infected during thefirst year of life do so.

[0006] Hepatitis B virus (HBV) infects over 300 million people worldwide(Imperial, 1999, Gastroenterol. Hepatol., 14 (suppl), S1-5). In theUnited States, approximately 1.25 million individuals are chroniccarriers of HBV as evidenced by the fact that they have measurablehepatitis B virus surface antigen HBsAg in their blood. The risk ofbecoming a chronic HBsAg carrier is dependent upon the mode ofacquisition of infection as well as the age of the individual at thetime of infection. For those individuals with high levels of viralreplication, chronic active hepatitis with progression to cirrhosis,liver failure and hepatocellular carcinoma (HCC) is common, and livertransplantation is the only treatment option for patients with end-stageliver disease from HBV.

[0007] The natural progression of chronic HBV infection over a 10 to 20year period leads to cirrhosis in 20-to-50% of patients and progressionof HBV infection to hepatocellular carcinoma has been well documented.There have been no studies that have determined sub-populations that aremost likely to progress to cirrhosis and/or hepatocellular carcinoma,thus all patients have equal risk of progression.

[0008] It is important to note that the survival for patients diagnosedwith hepatocellular carcinoma is only 0.9 to 12.8 months from initialdiagnosis (Takahashi et al., 1993, American Journal of Gastroenterology,88, 240-243). Treatment of hepatocellular carcinoma withchemotherapeutic agents has not proven effective and only 10% ofpatients will benefit from surgery due to extensive tumor invasion ofthe liver (Trinchet et al., 1994, Presse Medicine, 23, 831-833). Giventhe aggressive nature of primary hepatocellular carcinoma, the onlyviable treatment alternative to surgery is liver transplantation(Pichlmayr et al., 1994, Hepatology., 20, 33S-40S).

[0009] Upon progression to cirrhosis, patients with chronic HCVinfection present with clinical features, which are common to clinicalcirrhosis regardless of the initial cause (D'Amico et al., 1986,Digestive Diseases and Sciences, 31, 468-475). These clinical featuresmay include: bleeding esophageal varices, ascites, jaundice, andencephalopathy (Zakim D, Boyer T D. Hepatology a textbook of liverdisease, Second Edition Volume 1. 1990 W. B. Saunders Company.Philadelphia). In the early stages of cirrhosis, patients are classifiedas compensated, meaning that although liver tissue damage has occurred,the patient's liver is still able to detoxify metabolites in theblood-stream. In addition, most patients with compensated liver diseaseare asymptomatic and the minority with symptoms report only minorsymptoms such as dyspepsia and weakness. In the later stages ofcirrhosis, patients are classified as decompensated meaning that theirability to detoxify metabolites in the bloodstream is diminished and itis at this stage that the clinical features described above willpresent.

[0010] In 1986, D'Amico et al. described the clinical manifestations andsurvival rates in 1155 patients with both alcoholic and viral associatedcirrhosis (D'Amico supra). Of the 1155 patients, 435 (37%) hadcompensated disease although 70% were asymptomatic at the beginning ofthe study. The remaining 720 patients (63%) had decompensated liverdisease with 78% presenting with a history of ascites, 31% withjaundice, 17% had bleeding and 16% had encephalopathy. Hepatocellularcarcinoma was observed in six (0.5%) patients with compensated diseaseand in 30 (2.6%) patients with decompensated disease.

[0011] Over the course of six years, the patients with compensatedcirrhosis developed clinical features of decompensated disease at a rateof 10% per year. In most cases, ascites was the first presentation ofdecompensation. In addition, hepatocellular carcinoma developed in 59patients who initially presented with compensated disease by the end ofthe six-year study.

[0012] With respect to survival, the D'Amico study indicated that thefive-year survival rate for all patients on the study was only 40%. Thesix-year survival rate for the patients who initially had compensatedcirrhosis was 54% while the six-year survival rate for patients whoinitially presented with decompensated disease was only 21%. There wereno significant differences in the survival rates between the patientswho had alcoholic cirrhosis and the patients with viral relatedcirrhosis. The major causes of death for the patients in the D'Amicostudy were liver failure in 49%; hepatocellular carcinoma in 22%; and,bleeding in 13% (D'Amico supra).

[0013] Hepatitis B virus is a double-stranded circular DNA virus. It isa member of the Hepadnaviridae family. The virus consists of a centralcore that contains a core antigen (HBcAg) surrounded by an envelopecontaining a surface protein/surface antigen (HBsAg) and is 42 nm indiameter. It also contains an e antigen (HBeAg), which, along with HBcAgand HBsAg, is helpful in identifying this disease

[0014] In HBV virions, the genome is found in an incompletedouble-stranded form. HBV uses a reverse transcriptase to transcribe apositive-sense full length RNA version of its genome back into DNA. Thisreverse transcriptase also contains DNA polymerase activity and thusbegins replicating the newly synthesized minus-sense DNA strand.However, it appears that the core protein encapsidates thereverse-transcriptase/polymerase before it completes replication.

[0015] From the free-floating form, the virus must first attach itselfspecifically to a host cell membrane. Viral attachment is one of thecrucial steps that determines host and tissue specificity. However,currently there are no in vitro cell-lines that can be infected by HBV.There are some cells lines, such as HepG2, which can support viralreplication only upon transient or stable transfection using HBV DNA.

[0016] After attachment, fusion of the viral envelope and host membranemust occur to allow the viral core proteins containing the genome andpolymerase to enter the cell. Once inside, the genome is translocated tothe nucleus where it is repaired and cyclized.

[0017] The complete closed circular DNA genome of HBV remains in thenucleus and gives rise to four transcripts. These transcripts initiateat unique sites but share the same 3′-ends. The 3.5-kb pregenomic RNAserves as a template for reverse transcription and also encodes thenucleocapsid protein and polymerase. A subclass of this transcript witha 5′-end extension codes for the precore protein that, after processing,is secreted as HBV e antigen. The 2.4-kb RNA encompasses the pre-S1 openreading frame (ORF) that encodes the large surface protein. The 2.1-kbRNA encompasses the pre-S2 and S ORFs that encode the middle and smallsurface proteins, respectively. The smallest transcript (˜0.8-kb) codesfor the X protein, a transcriptional activator.

[0018] Multiplication of the HBV genome begins within the nucleus of aninfected cell. RNA polymerase II transcribes the circular HBV DNA intogreater-than-full length mRNA. Since the mRNA is longer than the actualcomplete circular DNA, redundant ends are formed. Once produced, thepregenomic RNA exits the nucleus and enters the cytoplasm.

[0019] The packaging of pregenomic RNA into core particles is triggeredby the binding of the HBV polymerase to the 5′ epsilon stem-loop. RNAencapsidation is believed to occur as soon as binding occurs. The HBVpolymerase also appears to require associated core protein in order tofunction. The HBV polymerase initiates reverse transcription from the 5′epsilon stem-loop three to four base pairs at which point the polymeraseand attached nascent DNA are transferred to the 3′ copy of the DR1region. Once there, the (−)DNA is extended by the HBV polymerase whilethe RNA template is degraded by the HBV polymerase RNAse H activity.When the HBV polymerase reaches the 5′ end, a small stretch of RNA isleft undigested by the RNAse H activity. This segment of RNA iscomprised of a small sequence just upstream and including the DR1region. The RNA oligomer is then translocated and annealed to the DR2region at the 5′ end of the (−)DNA. It is used as a primer for the(+)DNA synthesis which is also generated by the HBV polymerase. Itappears that the reverse transcription as well as plus strand synthesismay occur in the completed core particle.

[0020] Since the pregenomic RNA is required as a template for DNAsynthesis, this RNA is an excellent target for nucleic acid basedtherapeutics. Nucleoside analogues that have been documented to modulateHBV replication target the reverse transcriptase activity needed toconvert the pregenomic RNA into DNA. Nucleic acid decoy and aptamermodulation of HBV reverse transcriptase would be expected to result in asimilar modulation of HBV replication.

[0021] Therapeutic Approaches

[0022] Current therapeutic goals of treatment are three-fold: toeliminate infectivity and transmission of HBV to others, to arrest theprogression of liver disease and improve the clinical prognosis, and toprevent the development of hepatocellular carcinoma (HCC).

[0023] Interferon alpha use is the most common therapy for HBV; however,recently Lamivudine (3TC®) has been approved by the FDA. Interferonalpha (IFN-alpha) is one treatment for chronic hepatitis B. The standardduration of IFN-alpha therapy is 16 weeks, however, the optimaltreatment length is still poorly defined. A complete response (HBV DNAnegative HBeAg negative) occurs in approximately 25% of patients.Several factors have been identified that predict a favorable responseto therapy including: High ALT, low HBV DNA, being female, andheterosexual orientation.

[0024] There is also a risk of reactivation of the hepatitis B viruseven after a successful response, this occurs in around 5% of respondersand normally occurs within 1 year.

[0025] Side effects resulting from treatment with type 1 interferons canbe divided into four general categories including: Influenza-likesymptoms, neuropsychiatric, laboratory abnormalities, and othermiscellaneous side effects. Examples of influenza-like symptoms include,fatigue, fever, myalgia, malaise, appetite loss, tachycardia, rigors,headache and arthralgias. The influenza-like symptoms are usuallyshort-lived and tend to abate after the first four weeks of dosing(Dusheiko et al., 1994, Journal of Viral Hepatitis, 1, 3-5).Neuropsychiatric side effects include irritability, apathy, moodchanges, insomnia, cognitive changes, and depression. Laboratoryabnormalities include the reduction of myeloid cells, includinggranulocytes, platelets and to a lesser extent, red blood cells. Thesechanges in blood cell counts rarely lead to any significant clinicalsequellae. In addition, increases in triglyceride concentrations andelevations in serum alanine and aspartate aminotransferase concentrationhave been observed. Finally, thyroid abnormalities have been reported.These thyroid abnormalities are usually reversible after cessation ofinterferon therapy and can be controlled with appropriate medicationwhile on therapy. Miscellaneous side effects include nausea, diarrhea,abdominal and back pain, pruritus, alopecia, and rhinorrhea. In general,most side effects will abate after 4 to 8 weeks of therapy (Dushieko etal, supra).

[0026] Lamivudine (3TC®) is a nucleoside analogue, which is a verypotent and specific inhibitor of HBV DNA synthesis. Lamivudine hasrecently been approved for the treatment of chronic Hepatitis B. Unliketreatment with interferon, treatment with 3TC® does not eliminate theHBV from the patient. Rather, viral replication is controlled andchronic administration results in improvements in liver histology inover 50% of patients. Phase III studies with 3TC®, showed that treatmentfor one year was associated with reduced liver inflammation and a delayin scarring of the liver. In addition, patients treated with Lamivudine(100 mg per day) had a 98 percent reduction in hepatitis B DNA and asignificantly higher rate of seroconversion, suggesting diseaseimprovements after completion of therapy. However, stopping of therapyresulted in a reactivation of HBV replication in most patients. Inaddition recent reports have documented 3TC® resistance in approximately30% of patients.

[0027] Current therapies for treating HBV infection, includinginterferon and nucleoside analogues, are only partially effective. Inaddition, drug resistance to nucleoside analogues is now emerging,making treatment of chronic Hepatitis B more difficult. Thus, a needexists for effective treatment of this disease that utilizes antiviralmodulators that work by mechanisms other than those currently utilizedin the treatment of both acute and chronic hepatitis B infections.

[0028] Draper, U.S. Pat. No. 6,017,756, describes the use of ribozymesfor the inhibition of Hepatitis B Virus.

[0029] Passman et al., 2000, Biochem. Biophys. Res. Commun., 268(3),728-733.; Gan et al., 1998, J. Med. Coll. PLA, 13(3), 157-159.; Li etal., 1999, Jiefangjun Yixue Zazhi, 24(2), 99-101; Putlitz et al., 1999,J. Virol., 73(7), 5381-5387.; Kim et al., 1999, Biochem. Biophys. Res.Commun., 257(3), 759-765.; Xu et al., 1998, Bingdu Xuebao, 14(4),365-369.; Welch et al., 1997, Gene Ther., 4(7), 736-743.; Goldenberg etal., 1997, International PCT publication No. WO 97/08309, Wands et al.,1997, J. of Gastroenterology and Hepatology, 12(suppl.), S354-S369.;Ruiz et al., 1997, BioTechniques, 22(2), 338-345.; Gan et al., 1996, J.Med. Coll. PLA, 11(3), 171-175.; Beck and Nassal, 1995, Nucleic AcidsRes., 23(24), 4954-62.; Goldenberg, 1995, International PCT publicationNo. WO 95/22600.; Xu et al., 1993, Bingdu Xuebao, 9(4), 331-6.; Wang etal., 1993, Bingdu Xuebao, 9(3), 278-80, all describe ribozymes that aretargeted to cleave a specific HBV target site.

[0030] Hunt et al., U.S. Pat. No. 5,859,226, describes specificnon-naturally occurring oligonucleotide decoys intended to inhibit theexpression of MHC-II genes through binding of the RF-X transcriptionfactor, that can inhibit the expression of certain HBV and CMV viralproteins.

[0031] Kao et al., International PCT Publication No. WO 00/04141,describes linear single stranded nucleic acid molecules capable ofspecifically binding to viral polymerases and inhibiting the activity ofthe viral polymerase.

[0032] Lu, International PCT Publication No. WO 99/20641, describesspecific triplex-forming oligonucleotides used in treating HBVinfection.

SUMMARY OF THE INVENTION

[0033] This invention relates to nucleic acid molecules directed todisrupt the function of HBV reverse transcriptase. The invention alsorelates to nucleic acid molecules directed to disrupt the function ofthe Enhancer I core region of the HBV genomic DNA. In particular, thepresent invention describes the selection and function of nucleic acidmolecules, such as decoys and aptamers, capable of specifically bindingto the HBV reverse transcriptase (pol) primer and modulating reversetranscription of the HBV pregenomic RNA. In another embodiment, thepresent invention relates to nucleic acid molecules, such as decoys,antisense and aptamers, capable of specifically binding to the HBVreverse transcriptase (pol) and modulating reverse transcription of theHBV pregenomic RNA. In yet another embodiment, the present inventionrelates to nucleic acid molecules capable of specifically binding to theHBV Enhancer I core region and modulating transcription of the HBVgenomic DNA. The invention further relates to allosteric enzymaticnucleic acid molecules or “allozymes” that are used to modulate HBV geneexpression. Such allozymes are active in the presence of HBV-derivednucleic acids, peptides, and/or proteins such as HBV reversetranscriptase and/or a HBV reverse transcriptase primer sequence,thereby allowing the allozyme to selectively cleave a sequence of HBVDNA or RNA. Allozymes of the invention are also designed to be active inthe presence of HBV Enhancer I sequences and/or mutant HBV Enhancer Isequences, thereby allowing the allozyme to selectively cleave asequence of HBV DNA or RNA. These nucleic acid molecules can be used totreat diseases and disorders associated with HBV infection.

[0034] In one embodiment, the invention features a nucleic acid decoymolecule that specifically binds the hepatitis B virus (HBV) reversetranscriptase primer sequence. In another embodiment, the inventionfeatures a nucleic acid decoy molecule that specifically binds thehepatitis B virus (HBV) reverse transcriptase. In yet anotherembodiment, the invention features a nucleic acid decoy molecule thatspecifically binds to the HBV Enhancer I core sequence.

[0035] In one embodiment, the invention features a nucleic acid aptamerthat specifically binds the hepatitis B virus (HBV) reversetranscriptase primer. In another embodiment, the invention features anucleic acid aptamer that specifically binds the hepatitis B virus (HBV)reverse transcriptase. In yet another embodiment, the invention featuresa nucleic acid aptamer molecule that specifically binds to the HBVEnhancer I core sequence.

[0036] In one embodiment, the invention features an allozyme thatspecifically binds the hepatitis B virus (HBV) reverse transcriptaseprimer. In another embodiment, the invention features an allozyme thatspecifically binds the hepatitis B virus (HBV) reverse transcriptase. Inyet another embodiment, the invention features an allozyme thatspecifically binds to the HBV Enhancer I core sequence.

[0037] In yet another embodiment, the invention features a nucleic acidmolecule, for example a triplex forming nucleic acid molecule orantisense nucleic acid molecule, that binds the hepatitis B virus (HBV)reverse transcriptase primer. In another embodiment, the inventionfeatures a triplex forming nucleic acid molecule or antisense nucleicacid molecule that specifically binds the hepatitis B virus (HBV)reverse transcriptase. In yet another embodiment, the invention featuresa triplex forming nucleic acid molecule or antisense nucleic acidmolecule that specifically binds to the HBV Enhancer I core sequence.

[0038] In another embodiment, a nucleic acid molecule of the inventionbinds to Hepatocyte Nuclear Factor 3 (HNF3) and/or Hepatocyte NuclearFactor 4 (HNF4) binding sequence within the HBV Enhancer I region of HBVgenomic DNA, for example the plus strand and/or minus strand DNA of theEnhancer I region, and blocks the binding of HNF3 and/or HNF4 to theEnhancer 1 region.

[0039] In another embodiment, the nucleic acid molecule of the inventioncomprises a sequence having (UUCA)n domain, where n is an integer from1-10. In another embodiment, the nucleic acid molecules of the inventioncomprise the sequence of SEQ. ID NOs: 1-128.

[0040] In another embodiment, the invention features a pharmaceuticalcomposition comprising a nucleic acid molecule of the invention in apharmaceutically acceptable carrier. In another embodiment, theinvention features a mammalian cell, for example a human cell, includinga nucleic acid molecule contemplated by the invention.

[0041] In one embodiment, the invention features a method for treatmentof HBV infection, cirrhosis, liver failure, or hepatocellular carcinoma,comprising administering to a subject a nucleic acid molecule of theinvention under conditions suitable for the treatment.

[0042] In another embodiment, the invention features a method oftreatment of a subject having a condition associated with HBV infection,comprising contacting cells of said subject with a nucleic acid moleculeof the invention under conditions suitable for such treatment. Inanother embodiment, the invention features a method of treatment of asubject having a condition associated with HBV infection, comprisingcontacting cells of said subject with a nucleic acid molecule of theinvention, and further comprising the use of one or more drug therapies,for example type I interferon or 3TC® (lamivudine), under conditionssuitable for said treatment. In another embodiment, the other therapy isadministered simultaneously with or separately from the nucleic acidmolecule.

[0043] In another embodiment, the invention features a method formodulating HBV replication in a mammalian cell comprising administeringto the cell a nucleic acid molecule of the invention under conditionssuitable for the modulation.

[0044] In yet another embodiment, the invention features a method ofmodulating HBV reverse transcriptase activity comprising contacting HBVreverse transcriptase with a nucleic acid molecule of the invention, forexample a decoy or aptamer, under conditions suitable for the modulationof the HBV reverse transcriptase activity.

[0045] In another embodiment, the invention features a method ofmodulating HBV transcription, comprising contacting an HBV Enhancer Isequence with a nucleic molecule of the invention with under conditionssuitable for the modulation of HBV transcription.

[0046] In one embodiment, a nucleic acid molecule of the invention, forexample a decoy or aptamer, is chemically synthesized. In anotherembodiment, the nucleic acid molecule of the invention comprises atleast one nucleic acid sugar modification. In yet another embodiment,the nucleic acid molecule of the invention comprises at least onenucleic acid base modification. In another embodiment, the nucleic acidmolecule of the invention comprises at least one nucleic acid backbonemodification.

[0047] In another embodiment, the nucleic acid molecule of the inventioncomprises at least one 2′-O-alkyl, 2′-alkyl, 2′-alkoxylalkyl,2′-alkylthioalkyl, 2′-amino, 2′-O-amino, or 2′-halo modification and/orany combination thereof with or without 2′-deoxy and/or 2′-ribonucleotides. In yet another embodiment, the nucleic acid molecule of theinvention comprises all 2′-O-alkyl nucleotides, for example, all2′-O-allyl nucleotides.

[0048] In one embodiment, the nucleic acid molecule of the inventioncomprises a 5′-cap, 3′-cap, or 5′-3′ cap structure, for example anabasic or inverted abasic moiety.

[0049] In another embodiment, the nucleic acid molecule of the inventionis a linear nucleic acid molecule. In another embodiment, the nucleicacid molecule of the invention is a linear nucleic acid molecule thatcan optionally form a hairpin, loop, stem-loop, or other secondarystructure. In yet another embodiment, the nucleic acid molecule of theinvention is a circular nucleic acid molecule.

[0050] In one embodiment, the nucleic acid molecule of the invention isa single stranded oligonucleotide. In another embodiment, the nucleicacid molecule of the invention is a double-stranded oligonucleotide.

[0051] In one embodiment, the nucleic acid molecule of the inventioncomprises an oligonucleotide having about 3 to about 100 nucleotides. Inanother embodiment, the nucleic acid molecule of the invention comprisesan oligonucleotide having about 3 to about 24 nucleotides. In anotherembodiment, the nucleic acid molecule of the invention comprises anoligonucleotide having about 4 to about 16 nucleotides.

[0052] In another embodiment, the nucleic acid molecule of the inventionbinds irreversibly to the HBV reverse transcriptase target, for example,by covalent attachment of the nucleic acid molecule to the reversetranscriptase primer sequence. The covalent attachment can beaccomplished by introducing chemical modifications into the sequence ofthe nucleic acid molecule (for example, decoy or aptamer) that arecapable of forming covalent bonds with the reverse transcriptase primersequence.

[0053] In another embodiment, the nucleic acid molecule of the inventionbinds irreversibly to the HBV Enhancer I sequence target, for example,by covalent attachment of the nucleic acid molecule to the HBV EnhancerI sequence. The covalent attachment can be accomplished by introducingchemical modifications into the sequence of the nucleic acidmoleculethat are capable of forming covalent bonds with the Enhancer Isequence.

[0054] In another embodiment, the type I interferon contemplated by theinvention is interferon alpha, interferon beta, consensus interferon,polyethylene glycol interferon, polyethylene glycol interferon alpha 2a,polyethylene glycol interferon alpha 2b, or polyethylene glycolconsensus interferon.

[0055] In one embodiment, the invention features a pharmaceuticalcomposition comprising type I interferon and a nucleic acid molecule ofthe invention in a pharmaceutically acceptable carrier.

[0056] In another embodiment, the invention features a method ofadministering a nucleic acid molecule of the invention to a cell, forexample a mammalian cell or human cell, comprising contacting the cellwith the nucleic acid molecule under conditions suitable for theadministration. The nucleic acid molecule can be administeredindependently or in conjunction with other therapeutic compounds, suchas type I interferon or 3TC® (lamivudine).

[0057] In yet another embodiment, the invention features a method ofadministering a nucleic acid molecule of the invention to a cell, forexample a mammalian cell or human cell, comprising contacting the cellwith the nucleic acid molecule of the invention under conditionssuitable for the administration. independently or in conjunction withother therapeutic compounds, such as enzymatic nucleic acid molecules,antisense molecules, triplex forming oligonucleotides, 2,5-A chimeras,and/or RNAi.

[0058] In another embodiment, a nucleic acid molecule of the inventionis administered to a cell or subject in the presence of a deliveryreagent, for example a lipid, cationic lipid, phospholipid, or liposome.

[0059] In one embodiment, the invention features novel nucleicacid-based molecules and techniques, such as nucleic acid decoymolecules and/or aptamers, used alone or in combination with enzymaticnucleic acid molecules, antisense molecules, and/or RNAi, and methodsfor use to down regulate or modulate the expression of HBV RNA and/orreplication of HBV.

[0060] In another embodiment, the invention features the use of one ormore of the nucleic acid-based molecules and techniques to modulate theexpression of genes encoding HBV viral proteins. Specifically, theinvention features the use of nucleic acid-based molecules andtechniques to specifically modulate the expression of the HBV viralgenome.

[0061] In another embodiment, the invention features the use of one ormore of the nucleic acid-based molecules and techniques to modulate theactivity, expression, or level of cellular proteins required for HBVreplication. For example, the invention features the use of nucleicacid-based molecules and techniques to specifically modulate theactivity of cellular proteins required for HBV replication.

[0062] In another embodiment, the invention features a nucleic acidsensor molecule having an enzymatic nucleic acid domain and a sensordomain that interacts with an HBV peptide, protein, or polynucleotidesequence, for example, HBV reverse transcriptase, HBV reversetranscriptase primer, or the Enhancer I element of the HBV pregenomicRNA, wherein such interaction results in modulation of the activity ofthe enzymatic nucleic acid domain of the nucleic acid sensor molecule.The invention features HBV-specific nucleic acid sensor molecules orallozymes, and methods for their use to down regulate or modulate theexpression of HBV RNA capable of progression and/or maintenance ofhepatitis, hepatocellular carcinoma, cirrhosis, and/or liver failure. Inone embodiment, the enzymatic nucleic acid domain of a nucleic acidsensor molecule of the invention is a Hammerhead, Inozyme, G-cleaver,DNAzyme, Zinzyme, Amberzyme, or Hairpin enzymatic nucleic acid molecule.

[0063] In one embodiment, one or more nucleic acid molecules of theinvention are used to treat HBV-infected cells or an HBV-infectedsubject wherein the HBV is resistant or the subject does not respond totreatment with 3TC® (Lamivudine). The nucleic molecule(s) can be usedeither alone or in combination with other therapies under conditionssuitable for the treatment.

[0064] In another embodiment, one or more nucleic acid molecules of theinvention are used to treat HBV-infected cells or an HBV-infectedsubject, wherein the HBV is resistant or the subject does not respond totreatment with Interferon, for example Infergen®. The nucleicmolecule(s) can be used either alone or in combination with othertherapies under conditions suitable for the treatment.

[0065] By “modulate” is meant that the expression of the gene, or levelof RNA molecule or equivalent RNA molecules encoding one or moreproteins or protein subunits, or activity of one or more proteins orprotein subunits is up regulated or down regulated, such thatexpression, level, or activity is greater than or less than thatobserved in the absence of the modulator. For example, the term“modulate” can mean “inhibit,” but the use of the word “modulate” is notlimited to this definition.

[0066] By “inhibit” it is meant that the activity of HBV, HBV reversetranscriptase, or level of RNAs or equivalent RNAs encoding one or moreprotein subunits of HBV is reduced below that observed in the absence ofthe nucleic acid of the invention. In one embodiment, inhibition withnucleic acid decoy molecule preferably is below that level observed inthe presence of an inactive or attenuated molecule that is unable tobind to the same site on the viral polymerase. In another embodiment,inhibition of HBV reverse transcriptase with the nucleic acid moleculeof the instant invention is greater in the presence of the nucleic acidmolecule than in its absence.

[0067] The methods of this invention can be used to treat humanhepatitis B virus infections, which include, for example, productivevirus infection, latent or persistent virus infection, and HBV-inducedhepatocyte transformation. The utility can be extended to other speciesof HBV that infect non-human animals where such infections are ofveterinary importance.

[0068] Preferred binding sites of the nucleic acid molecules of theinvention include, but are not limited to, the primer binding site onHBV reverse transcriptase, the primer binding sequences of the HBV RNA,and/or the HBV Enhancer I region of HBV DNA.

[0069] By “nucleic acid decoy molecule” or “decoy” as used herein ismeant a nucleic acid molecule that mimics the natural binding domain fora ligand. The decoy therefore competes with the natural binding targetfor the binding of a specific ligand. For example, it has been shownthat over-expression of HIV trans-activation response (TAR) RNA can actas a “decoy” and efficiently binds HIV tat protein, thereby preventingit from binding to TAR sequences encoded in the HIV RNA (Sullenger etal., 1990, Cell, 63, 601-608). Similarly, the nucleic acid molecules ofthe instant invention can bind to HBV reverse transcriptase, HBV reversetranscriptase primer, or Enhancer I element of the HBV pregenomic RNA toblock transcription of the HBV genome.

[0070] By “aptamer” or “nucleic acid aptamer” as used herein is meant anucleic acid molecule that binds specifically to a target moleculewherein the nucleic acid molecule has sequence that is distinct fromsequence recognized by the target molecule in its natural setting.Alternately, an aptamer can be a nucleic acid molecule that binds to atarget molecule where the target molecule does not naturally bind to anucleic acid. The target molecule can be any molecule of interest. Forexample, the aptamer can be used to bind to a ligand-binding domain of aprotein, thereby preventing interaction of the naturally occurringligand with the protein. This is a non-limiting example and those in theart will recognize that other embodiments can be readily generated usingtechniques generally known in the art, see for example Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.

[0071] By “enzymatic nucleic acid molecule” is meant a nucleic acidmolecule that has complementarity in a substrate binding region to aspecified gene target, and also has an enzymatic activity which isactive to specifically cleave a target RNA molecule. That is, theenzymatic nucleic acid molecule is able to intermolecularly cleave a RNAmolecule and thereby inactivate a target RNA molecule. Thesecomplementary regions allow sufficient hybridization of the enzymaticnucleic acid molecule to a target RNA molecule and thus permit cleavage.One hundred percent complementarity is preferred, but complementarity aslow as 50-75% can also be useful in this invention (see for exampleWerner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096;Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31).The nucleic acids can be modified at the base, sugar, and/or phosphategroups. The term enzymatic nucleic acid is used interchangeably withphrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA,aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalyticoligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of theseterminologies describe nucleic acid molecules with enzymatic activity.The specific enzymatic nucleic acid molecules described in the instantapplication are not limiting in the invention and those skilled in theart will recognize that all that is important in an enzymatic nucleicacid molecule of this invention is that it have a specific substratebinding site which is complementary to one or more of the target nucleicacid regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart a nucleic acidcleaving activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071;Cech et al., 1988, JAMA 260:20 3030-4).

[0072] By “nucleic acid molecule” as used herein is meant a moleculecomprising nucleotides. The nucleic acid can be single, double, ormultiple stranded and can comprise modified or unmodified nucleotides ornon-nucleotides or various mixtures and combinations thereof.

[0073] By “Inozyme” or “NCH” motif or configuration is meant, anenzymatic nucleic acid molecule comprising a motif as is generallydescribed as NCH Rz in Ludwig et al., International PCT Publication No.WO 98/58058 and U.S. patent application Ser. No. 08/878,640. Inozymespossess endonuclease activity to cleave RNA substrates having a cleavagetriplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine,uridine or cytidine, and/represents the cleavage site. Inozymes can alsopossess endonuclease activity to cleave RNA substrates having a cleavagetriplet NCN/, where N is a nucleotide, C is cytidine, and/represents thecleavage site

[0074] By “G-cleaver” motif or configuration is meant, an enzymaticnucleic acid molecule comprising a motif as is generally described inEckstein et al., U.S. Pat. No. 6,127,173 and in Kore et al., 1998,Nucleic Acids Research 26, 4116-4120. G-cleavers possess endonucleaseactivity to cleave RNA substrates having a cleavage triplet NYN/, whereN is a nucleotide, Y is uridine or cytidine and/represents the cleavagesite. G-cleavers can be chemically modified.

[0075] By “zinzyme” motif or configuration is meant, an enzymaticnucleic acid molecule comprising a motif as is generally described inBeigelman et al., International PCT publication No. WO 99/55857 and U.S.patent application Ser. No. 09/918,728. Zinzymes possess endonucleaseactivity to cleave RNA substrates having a cleavage triplet includingbut not limited to, YG/Y, where Y is uridine or cytidine, and G isguanosine and/represents the cleavage site. Zinzymes can be chemicallymodified to increase nuclease stability through various substitutions,including substituting 2′-O-methyl guanosine nucleotides for guanosinenucleotides. In addition, differing nucleotide and/or non-nucleotidelinkers can be used to substitute the 5′-gaaa-2′ loop of the motif.Zinzymes represent a non-limiting example of an enzymatic nucleic acidmolecule that does not require a ribonucleotide (2′-OH) group within itsown nucleic acid sequence for activity.

[0076] By “amberzyme” motif or configuration is meant, an enzymaticnucleic acid molecule comprising a motif as is generally described inBeigelman et al., International PCT publication No. WO 99/55857 and U.S.patent application Ser. No. 09/476,387. Amberzymes possess endonucleaseactivity to cleave RNA substrates having a cleavage triplet NG/N, whereN is a nucleotide, G is guanosine, and/represents the cleavage site.Amberzymes can be chemically modified to increase nuclease stability. Inaddition, differing nucleoside and/or non-nucleoside linkers can be usedto substitute the 5′-gaaa-3′ loops of the motif. Amberzymes represent anon-limiting example of an enzymatic nucleic acid molecule that does notrequire a ribonucleotide (2′-OH) group within its own nucleic acidsequence for activity.

[0077] By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule thatdoes not require the presence of a 2′-OH group within its own nucleicacid sequence for activity. In particular embodiments, the enzymaticnucleic acid molecule can have an attached linker or linkers or otherattached or associated groups, moieties, or chains containing one ormore nucleotides with 2′-OH groups. DNAzymes can be synthesizedchemically or expressed endogenously in vivo, by means of a singlestranded DNA vector or equivalent thereof. Non-limiting examples ofDNAzymes are generally reviewed in Usman et al., U.S. Pat. No.6,159,714; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995,Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999,Nature Biotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am.Chem. Soc., 122, 2433-39. The “10-23” DNAzyme motif is one particulartype of DNAzyme that was evolved using in vitro selection as generallydescribed in Joyce et al., U.S. Pat. No. 5,807,718 and Santoro et al.,supra. Additional DNAzyme motifs can be selected for using techniquessimilar to those described in these references, and hence, are withinthe scope of the present invention.

[0078] By “nucleic acid sensor molecule” or “allozyme” as used herein ismeant a nucleic acid molecule comprising an enzymatic domain and asensor domain, where the enzymatic nucleic acid domain's ability tocatalyze a chemical reaction is dependent on the interaction with atarget signaling molecule, such as a nucleic acid, polynucleotide,oligonucleotide, peptide, polypeptide, or protein, for example HBV RT,HBV RT primer, or HBV Enhancer I sequence. The introduction of chemicalmodifications, additional functional groups, and/or linkers, to thenucleic acid sensor molecule can provide enhanced catalytic activity ofthe nucleic acid sensor molecule, increased binding affinity of thesensor domain to a target nucleic acid, and/or improvednuclease/chemical stability of the nucleic acid sensor molecule, and arehence within the scope of the present invention (see for example Usmanet al., U.S. patent application Ser. No. 09/877,526, George et al., U.S.Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332,Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington,International PCT publication No. WO 00/24931, Breaker et al.,International PCT Publication Nos. WO 00/26226 and 98/27104, andSullenger et al., U.S. patent application Ser. No. 09/205,520).

[0079] By “sensor component” or “sensor domain” of the nucleic acidsensor molecule as used herein is meant, a nucleic acid sequence (e.g.,RNA or DNA or analogs thereof) which interacts with a target signalingmolecule, for example a nucleic acid sequence in one or more regions ofa target nucleic acid molecule or more than one target nucleic acidmolecule, and which interaction causes the enzymatic nucleic acidcomponent of the nucleic acid sensor molecule to either catalyze areaction or stop catalyzing a reaction. In the presence of a targetsignaling molecule of the invention, such as HBV RT, HBV RT primer, orHBV Enhancer I sequence, the ability of the sensor component, forexample, to modulate the catalytic activity of the nucleic acid sensormolecule, is modulated or diminished. The sensor component can compriserecognition properties relating to chemical or physical signals capableof modulating the nucleic acid sensor molecule via chemical or physicalchanges to the structure of the nucleic acid sensor molecule. The sensorcomponent can be derived from a naturally occurring nucleic acid bindingsequence, for example, RNAs that bind to other nucleic acid sequences invivo. Alternately, the sensor component can be derived from a nucleicacid molecule (aptamer), which is evolved to bind to a nucleic acidsequence within a target nucleic acid molecule. The sensor component canbe covalently linked to the nucleic acid sensor molecule, or can benon-covalently associated. A person skilled in the art will recognizethat all that is required is that the sensor component is able toselectively modulate the activity of the nucleic acid sensor molecule tocatalyze a reaction.

[0080] By “target molecule” or “target signaling molecule” is meant amolecule capable of interacting with a nucleic acid sensor molecule,specifically a sensor domain of a nucleic acid sensor molecule, in amanner that causes the nucleic acid sensor molecule to be active orinactive. The interaction of the signaling agent with a nucleic acidsensor molecule can result in modification of the enzymatic nucleic acidcomponent of the nucleic acid sensor molecule via chemical, physical,topological, or conformational changes to the structure of the molecule,such that the activity of the enzymatic nucleic acid component of thenucleic acid sensor molecule is modulated, for example is activated ordeactivated. Signaling agents can comprise target signaling moleculessuch as macromolecules, ligands, small molecules, metals and ions,nucleic acid molecules including but not limited to RNA and DNA oranalogs thereof, proteins, peptides, antibodies, polysaccharides,lipids, sugars, microbial or cellular metabolites, pharmaceuticals, andorganic and inorganic molecules in a purified or unpurified form, forexample HBV RT or HBV RT primer.

[0081] By “sufficient length” is meant a nucleic acid molecule longenough to provide the intended function under the expected condition.For example, a nucleic acid molecule of the invention needs to be of“sufficient length” to provide stable binding to a target site under theexpected binding conditions and environment. In another non-limitingexample, for the binding arms of an enzymatic nucleic acid, “sufficientlength” means that the binding arm sequence is long enough to providestable binding to a target site under the expected reaction conditionsand environment. The binding arms are not so long as to prevent usefulturnover of the nucleic acid molecule. By “stably interact” is meantinteraction of the oligonucleotides with target nucleic acid (e.g., byforming hydrogen bonds with complementary nucleotides in the targetunder physiological conditions) that is sufficient for the intendedpurpose (e.g., cleavage of target RNA by an enzyme).

[0082] By “homology” is meant the nucleotide sequence of two or morenucleic acid molecules is partially or completely identical.

[0083] By “antisense nucleic acid”, it is meant a non-enzymatic nucleicacid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA orRNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566)interactions and alters the activity of the target RNA (for a review,see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al, U.S. Pat.No. 5,849,902). Typically, antisense molecules are complementary to atarget sequence along a single contiguous sequence of the antisensemolecule. However, in certain embodiments, an antisense molecule canbind to substrate such that the substrate molecule forms a loop, and/oran antisense molecule can bind such that the antisense molecule forms aloop. Thus, the antisense molecule can be complementary to two or morenon-contiguous substrate sequences or two or more non-contiguoussequence portions of an antisense molecule can be complementary to atarget sequence, or both. For a review of current antisense strategies,see Schmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas etal., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. DrugDev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998,Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol.,40, 1-49. Antisense molecules of the instant invention can include 2-5Aantisense chimera molecules. In addition, antisense DNA can be used totarget RNA by means of DNA-RNA interactions, thereby activating RNase H,which digests the target RNA in the duplex. The antisenseoligonucleotides can comprise one or more RNAse H activating region thatis capable of activating RNAse H cleavage of a target RNA. Antisense DNAcan be synthesized chemically or expressed via the use of a singlestranded DNA expression vector or equivalent thereof.

[0084] By “RNase H activating region” is meant a region (generallygreater than or equal to 4-25 nucleotides in length, preferably from5-11 nucleotides in length) of a nucleic acid molecule capable ofbinding to a target RNA to form a non-covalent complex that isrecognized by cellular RNase H enzyme (see for example Arrow et al.,U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). TheRNase H enzyme binds to the nucleic acid molecule-target RNA complex andcleaves the target RNA sequence. The RNase H activating regioncomprises, for example, phosphodiester, phosphorothioate (for example,at least four of the nucleotides are phosphorothiote substitutions; morespecifically, 4-11 of the nucleotides are phosphorothiotesubstitutions), phosphorodithioate, 5′-thiophosphate, ormethylphosphonate backbone chemistry or a combination thereof. Inaddition to one or more backbone chemistries described above, the RNaseH activating region can also comprise a variety of sugar chemistries.For example, the RNase H activating region can comprise deoxyribose,arabino, fluoroarabino or a combination thereof, nucleotide sugarchemistry. Those skilled in the art will recognize that the foregoingare non-limiting examples and that any combination of phosphate, sugarand base chemistry of a nucleic acid that supports the activity of RNaseH enzyme is within the scope of the definition of the RNase H activatingregion and the instant invention.

[0085] By “2-SA antisense chimera” it is meant, an antisenseoligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylateresidue. These chimeras bind to target RNA in a sequence-specific mannerand activate a cellular 2-SA-dependent ribonuclease, which, in turn,cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA90, 1300).

[0086] By “triplex nucleic acid” or “triplex oligonucleotide” it ismeant a polynucleotide or oligonucleotide that can bind to adouble-stranded DNA in a sequence-specific manner to form atriple-strand helix. Formation of such triple helix structure has beenshown to modulate transcription of the targeted gene (Duval-Valentin etal., 1992, Proc. Natl. Acad. Sci. USA, 89, 504). Triplex nucleic acidmolecules of the invention also include steric blocker nucleic acidmolecules that bind to the Enhancer I region of HBV DNA (plus strandand/or minus strand) and prevent translation of HBV genomic DNA.

[0087] The term “double stranded RNA” or “dsRNA” as used herein refersto a double stranded RNA molecule capable of RNA interference “RNAi”,including short interfering RNA “siRNA” see for example Bass, 2001,Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; andKreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914.

[0088] By “gene” it is meant a nucleic acid that encodes an RNA, forexample, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide.

[0089] By “complementarity” is meant that a nucleic acid can formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. In reference tothe nucleic molecules of the present invention, the binding free energyfor a nucleic acid molecule with its target or complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., ribozyme cleavage, antisense or triple helix modulation.Determination of binding free energies for nucleic acid molecules iswell known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant.Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Apercent complementarity indicates the percentage of contiguous residuesin a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.

[0090] The nucleic acid decoy molecules and/or aptamers that bind to areverse transcriptase and/or reverse transcriptase primer and thereforeinactivate the reverse transcriptase, represent a novel therapeuticapproach to treat a variety of pathologic indications, including, viralinfection such as HBV infection, hepatitis, hepatocellular carcinoma,tumorigenesis, cirrhosis, liver failure and others.

[0091] The nucleic acid molecules that bind to a HBV Enhancer I sequenceand therefore inactivate HBV transcription, represent a noveltherapeutic approach to treat a variety of pathologic indications,including viral infection, such as HBV infection, hepatitis,hepatocellular carcinoma, tumorigenesis, cirrhosis, liver failure andothers.

[0092] In one embodiment of the present invention, a decoy nucleic acidmolecule of the invention is 4 to 50 nucleotides in length, in specificembodiments about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16nucleotides in length. In another embodiment, a non-decoy nucleic acidmolecule, e.g., an antisense molecule, a triplex DNA, or a ribozyme, is13 to 100 nucleotides in length, e.g., in specific embodiments 35, 36,37, or 38 nucleotides in length (e.g., for particular ribozymes orantisense). In particular embodiments, the nucleic acid molecule is15-100, 17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100, 32-100,35-100, 40-100, 50-100, 60-100, 70-100, or 80-100 nucleotides in length.Instead of 100 nucleotides being the upper limit on the length rangesspecified above, the upper limit of the length range can be, forexample, 30, 40, 50, 60, 70, or 80 nucleotides. Thus, for any of thelength ranges, the length range for particular embodiments has lowerlimit as specified, with an upper limit as specified which is greaterthan the lower limit. For example, in a particular embodiment, thelength range can be 35-50 nucleotides in length. All such ranges areexpressly included. Also in particular embodiments, a nucleic acidmolecule can have a length which is any of the lengths specified above,for example, 21 nucleotides in length.

[0093] Exemplary nucleic acid decoy molecules of the invention are shownin Table I. Exemplary synthetic nucleic acid molecules of the inventionare shown in Table II. For example, decoy molecules of the invention areabout 4 to about 50 nucleotides in length. Exemplary decoys of theinvention are 4, 8, 12, or 16 nucleotides in length. In an additionalexample, enzymatic nucleic acid molecules of the invention are about 15to about 50 nucleotides in length, for example, 25 to 40 nucleotides inlength, e.g., 34, 36, or 38 nucleotides in length (for example seeJarvis et al., 1996, J. Biol. Chem., 271, 29107-29112). ExemplaryDNAzymes of the invention are about 15 to about 40 nucleotides inlength, for example, 25 to 35 nucleotides in length, e.g., 29, 30, 31,or 32 nucleotides in length (see for example Santoro et al., 1998,Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic AcidsResearch, 23, 4092-4096). Exemplary antisense molecules of the inventionare about 15 to about 75 nucleotides in length, for example, 20 to 35nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length(see for example Woolf et al., 1992, PNAS., 89, 7305-7309; Milner etal., 1997, Nature Biotechnology, 15, 537-541). Exemplary triplex formingoligonucleotide molecules of the invention are about 10 to about 40nucleotides in length, for example, 12 to 25 nucleotides in length,e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher etal., 1990, Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990,Science, 249, 73-75). Those skilled in the art will recognize that allthat is required is that the nucleic acid molecule is of length andconformation sufficient and suitable for the nucleic acid molecule tocatalyze a reaction contemplated herein. The length of the nucleic acidmolecules of the instant invention are not limiting within the generallimits stated.

[0094] In one embodiment, the invention provides a method for producinga class of nucleic acid-based gene modulating agents, which exhibit ahigh degree of specificity for a viral reverse transcriptase, such asHBV reverse transcriptase, or reverse transcriptase primer, such as aHBV reverse transcriptase primer. For example, the nucleic acid moleculecan be targeted to a highly conserved nucleic acid binding region of theviral reverse transcriptase such that specific treatment of a disease orcondition can be provided with either one or several nucleic acidmolecules of the invention. Such nucleic acid molecules can be deliveredexogenously to specific tissue or cellular targets as required.Alternatively, the nucleic acid molecules can be expressed from DNAand/or RNA vectors that are delivered to specific cells.

[0095] In another embodiment, the invention provides a method forproducing a class of nucleic acid-based gene modulating agents whichexhibit a high degree of specificity for a viral enhancer regions, suchas the HBV Enhancer I core sequence. For example, the nucleic acidmolecule can be targeted to a highly conserved transcriptionfactor-binding region of the viral Enhancer I sequence such thatspecific treatment of a disease or condition can be provided with eitherone or several nucleic acid molecules of the invention. Such nucleicacid molecules can be delivered exogenously to specific tissue orcellular targets as required. Alternatively, the nucleic acid moleculescan be expressed from DNA and/or RNA vectors that are delivered tospecific cells.

[0096] As used herein “cell” is used in its usual biological sense, anddoes not refer to an entire multicellular organism, e.g., specificallydoes not refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell).

[0097] By “HBV proteins” is meant, a protein or a mutant proteinderivative thereof, comprising sequence expressed and/or encoded by theHBV genome.

[0098] By “highly conserved nucleic acid binding region” is meant anamino acid sequence of one or more regions in a target protein that doesnot vary significantly from one generation to the other or from onebiological system to the other.

[0099] The enzymatic nucleic acid-based modulators of HBV expression areuseful for the prevention of the diseases and conditions including HBVinfection, hepatitis, cancer, cirrhosis, liver failure, and any otherdiseases or conditions that are related to the levels of HBV in a cellor tissue.

[0100] By “related to the levels of HBV” is meant that the reduction ofHBV expression or HBV RNA or protein levels will relieve, to someextent, the symptoms of the disease or condition.

[0101] The nucleic acid-based modulators of the invention are addeddirectly, or can be complexed with cationic lipids, packaged withinliposomes, or otherwise delivered to target cells or tissues. Thenucleic acid or nucleic acid complexes can be locally administered torelevant tissues ex vivo, or in vivo through injection, infusion pump orstent, with or without their incorporation in biopolymers. In particularembodiments, the nucleic acid molecules of the invention comprisesequences shown in Table I and Table II. Examples of such nucleic acidmolecules consist essentially of sequences defined in the tables.

[0102] In another aspect, the invention provides mammalian cellscomprising one or more nucleic acid molecules and/or expression vectorsof this invention. The one or more nucleic acid molecules canindependently be targeted to the same or different sites.

[0103] In another aspect of the invention, nucleic acid decoys,aptamers, enzymatic nucleic acids or antisense molecules that interactwith target protein and/or RNA molecules and modulate HBV (for example,HBV reverse transcriptase, or transcription of HBV genomic DNA) activityare expressed from transcription units inserted into DNA or RNA vectors.The recombinant vectors are preferably DNA plasmids or viral vectors.Decoys, aptamers, enzymatic nucleic acid or antisense expressing viralvectors can be constructed based on, but not limited to,adeno-associated virus, retrovirus, adenovirus, or alphavirus.Preferably, the recombinant vectors capable of expressing the decoys,aptamers, enzymatic nucleic acids or antisense are delivered asdescribed above, and persist in target cells. Alternatively, viralvectors can be used that provide for transient expression of decoys,aptamers, enzymatic nucleic acids or antisense. Such vectors might berepeatedly administered as necessary. Once expressed, the decoys,aptamers, enzymatic nucleic acids or antisense bind to the targetprotein and/or RNA and modulate its function or expression. Delivery ofdecoy, aptamer, enzymatic nucleic acid or antisense expressing vectorscan be systemic, such as by intravenous or intramuscular administration,by administration to target cells ex-planted from the subject followedby reintroduction into the subject, or by any other means that wouldallow for introduction into the desired target cell. DNA based nucleicacid molecules of the invention can be expressed via the use of a singlestranded DNA intracellular expression vector.

[0104] By RNA is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety.

[0105] By “vectors” is meant any nucleic acid- and/or viral-basedtechnique used to express and/or deliver a desired nucleic acid.

[0106] By “subject” is meant an organism, which is a donor or recipientof explanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. In one embodiment, a subject is a mammal or mammaliancells. In another embodiment, a subject is a human or human cells.

[0107] The nucleic acid molecules of the instant invention,individually, or in combination or in conjunction with other drugs, canbe used to treat diseases or conditions discussed herein. For example,to treat a disease or condition associated with the levels of HBV, thenucleic acid molecules can be administered to a subject or can beadministered to other appropriate cells evident to those skilled in theart, individually or in combination with one or more drugs underconditions suitable for the treatment.

[0108] In a further embodiment, the described molecules, such as decoys,aptamers, antisense, or enzymatic nucleic acids, can be used incombination with other known treatments to treat conditions or diseasesdiscussed above. For example, the described molecules can be used incombination with one or more known therapeutic agents to treat acondition related to HBV levels, such as HBV infection, hepatitis,hepatocellular carcinoma, cancer, cirrhosis, and liver failure. Suchtherapeutic agents include, but are not limited to, nucleoside analogsselected from the group comprising Lamivudine (3TC®), L-FMAU, and/oradefovir dipivoxil (for a review of applicable nucleoside analogs, seeColacino and Staschke, 1998, Progress in Drug Research, 50, 259-322).Immunomodulators selected from the group comprising Type 1 Interferon,therapeutic vaccines, steriods, and 2′-5′ oligoadenylates (for a reviewof 2′-5′ Oligoadenylates, see Charubala and Pfleiderer, 1994, Progressin Molecular and Subcellular Biology, 14, 113-138).

[0109] In another embodiment, the invention features nucleic acid-basedmodulators (e.g., nucleic acid decoy molecules, aptamers, enzymaticnucleic acid molecules (ribozymes), antisense nucleic acids, triplexDNA, antisense nucleic acids containing RNA cleaving chemical groups)and methods for their use to down regulate or modulate reversetranscriptase activity and/or the expression of RNA (e.g., HBV) capableof progression and/or maintenance of HBV infection, hepatocellularcarcinoma, liver disease and failure.

[0110] In another embodiment, the invention features nucleic acid-basedmolecules and techniques (e.g., nucleic acid decoy molecules, aptamers,enzymatic nuleic acid molecules (ribozymes), antisense nucleic acidmolecules, triplex DNA, antisense nucleic acids containing RNA cleavingchemical groups) and methods for their use to down regulate or modulatereverse transcriptase activity and/or the expression of HBV RNA.

[0111] In another embodiment, the invention features nucleic acid-basedmodulators (e.g., nucleic acid decoy molecules, aptamers, enzymaticnucleic acid molecules (ribozymes), antisense nucleic acids, triplexDNA, siRNA, dsRNA, antisense nucleic acids containing RNA cleavingchemical groups) and methods for their use to down regulate or modulateEnhancer I mediated transcription activity and/or the expression of DNA(e.g., HBV) capable of progression and/or maintenance of HBV infection,hepatocellular carcinoma, liver disease and failure.

[0112] In another embodiment, the invention features nucleic acid-basedmolecules and techniques (e.g., nucleic acid decoy molecules, aptamers,enzymatic nuleic acid molecules, antisense nucleic acid molecules,triplex DNA, antisense nucleic acids containing DNA cleaving chemicalgroups) and methods for their use to down regulate or modulate EnhancerI mediated transcription activity and/or the expression of HBV DNA.

[0113] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0114] First the drawings will be described briefly.

DRAWINGS

[0115]FIG. 1 is a schematic diagram which outlines the steps involved inHBV reverse transcription. The HBV polymerase/reverse transcriptasebinds to the 5′-stem-loop of the HBV pregenomic RNA and synthesizes aprimer from the UUCA template. The reverse transcriptase and tetramerprimer are translocated to the 3′-DR1 site. The RT primer binds to theUUCA sequence in the DR1 element and minus strand synthesis begins.

[0116]FIG. 2 is a schematic diagram showing a non-limiting example ofinhibition of HBV reverse transcription. In this example, a decoymolecule binds to the HBV RT primer, thereby preventing translocation ofthe RT to the 3′-DR1 site and preventing minus strand synthesis.

[0117]FIG. 3 is a graphical representation of HbsAg levels in thepresence of different 2′-O-allyl modified nucleic acid molecules asdetermined by ELISA. Inhibition of HBV is correlated to HBsAg antigenlevels.

[0118]FIG. 4 is a graphical representation of HbsAg levels in thepresence of different 2′-O-methyl modified nucleic acid molecules asdetermined by ELISA. Inhibition of HBV is correlated to HBsAg antigenlevels.

[0119]FIG. 5 is a graphical representation of HbsAg levels in thepresence of various concentrations (100-800 nM) of inverse controlnucleic acid molecules and 2′-O-methyl modified nucleic acid moleculestargeted to the HBV reverse transcriptase primer.

[0120]FIG. 6 is a graphical representation of HbsAg levels in thepresence of nucleic acid molecules (200 nM) targeted to the HBV EnhancerI core region.

[0121]FIG. 7 is a graphical representation of HbsAg levels in thepresence of nucleic acid molecules (400 nM) targeted to the HBV EnhancerI core region.

[0122]FIG. 8 is a graphical representation of HbsAg levels in thepresence of various concentrations (50-200 nM) of inverse controlnucleic acid molecules and nucleic acid molecules targeted to the HBVEnhancer I core region.

MECHANISM OF ACTION OF NUCLEIC ACID MOLECULES OF THE INVENTION

[0123] Decoy: Nucleic acid decoy molecules are mimetics of naturallyoccurring nucleic acid molecules or portions of naturally occurringnucleic acid molecules that can be used to modulate the function of aspecific protein or a nucleic acid whose activity is dependant oninteraction with the naturally occurring nucleic acid molecule. Decoysmodulate the function of a target protein or nucleic acid by competingwith authentic nucleic acid binding to the ligand of interest. Often,the nucleic acid decoy is a truncated version of a nucleic acid sequencethat is recognized, for example by a particular protein, such as atranscription factor or polymerase. Decoys can be chemically modified toincrease binding affinity to the target ligand as well as to increasethe enzymatic and chemical stability of the decoy. In addition, bridgingand non-bridging linkers can be introduced into the decoy sequence toprovide additional binding affinity to the target ligand. Decoymolecules of the invention that bind to a reverse transcriptase orreverse transcriptase primer, for example, HBV reverse transcriptase orHBV reverse transcriptase primer, or an enhancer region of the HBVpregenomic RNA, for example, the Enhancer I element, modulate thetranscription of RNA to DNA and therefore modulate expression of thepregenomic RNA of the virus (see FIGS. 1-2).

[0124] Aptamer: Nucleic acid aptamers can be selected to specificallybind to a particular ligand of interest (see for example Gold et al.,U.S. Pat. No. 5,567,588 and U.S. Pat. No. 5,475,096, Gold et al., 1995,Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74,5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J.Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; andJayasena, 1999, Clinical Chemistry, 45, 1628). For example, the use ofin vitro selection can be applied to evolve nucleic acid aptamers withbinding specificity for HBV RT and/or HBV RT primer. Nucleic acidaptamers can include chemical modifications and linkers as describedherein. Aptamer molecules of the invention that bind to a reversetranscriptase or reverse transcriptase primer, such as HBV reversetranscriptase or HBV reverse transcriptase primer, modulate thetranscription of RNA to DNA and therefore modulate expression of thepregenomic RNA of the virus.

[0125] Antisense: Antisense molecules can be modified or unmodified RNA,DNA, or mixed polymer oligonucleotides and primarily function byspecifically binding to matching sequences resulting in modulation ofpeptide synthesis (Wu-Pong, Nov 1994, BioPharm, 20-33). The antisenseoligonucleotide binds to target RNA by Watson Crick base-pairing andblocks gene expression by preventing ribosomal translation of the boundsequences either by steric blocking or by activating RNase H enzyme.Antisense molecules can also alter protein synthesis by interfering withRNA processing or transport from the nucleus into the cytoplasm(Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).

[0126] In addition, binding of single stranded DNA to RNA may result innuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke,supra). To date, the only backbone modified DNA chemistry which will actas substrates for RNase H are phosphorothioates, phosphorodithioates,and borontrifluoridates. Recently, it has been reported that 2′-arabinoand 2′-fluoro arabino-containing oligos can also activate RNase Hactivity.

[0127] A number of antisense molecules have been described that utilizenovel configurations of chemically modified nucleotides, secondarystructure, and/or RNase H substrate domains (Woolf et al., InternationalPCT Publication No. WO 98/13526; Thompson et al., U.S. S No. 60/082,404which was filed on Apr. 20, 1998; Hartmann et al., U.S. S No. 60/101,174which was filed on Sep. 21, 1998) all of these are incorporated byreference herein in their entirety.

[0128] Antisense DNA can be used to target RNA by means of DNA-RNAinteractions, thereby activating RNase H, which digests the target RNAin the duplex. Antisense DNA can be chemically synthesized or can beexpressed via the use of a single stranded DNA intracellular expressionvector or the equivalent thereof.

[0129] Triplex Forming Oligonucleotides (TFO): Single strandedoligonucleotide can be designed to bind to genomic DNA in a sequencespecific manner. TFOs can be comprised of pyrimidine-richoligonucleotides which bind DNA helices through Hoogsteen Base-pairing(Wu-Pong, supra). In addition, TFOs can be chemically modified toincrease binding affinity to target DNA sequences. The resulting triplehelix composed of the DNA sense, DNA antisense, and TFO disrupts RNAsynthesis by RNA polymerase. The TFO mechanism can result in geneexpression or cell death since binding may be irreversible (Mukhopadhyay& Roth, supra)

[0130] 2′-5′ Oligoadenylates: The 2-5A system is an interferon-mediatedmechanism for RNA degradation found in higher vertebrates (Mitra et al.,1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5Asynthetase and RNase L, are required for RNA cleavage. The 2-5Asynthetases require double stranded RNA to form 2′-5′ oligoadenylates(2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L,which has the ability to cleave single stranded RNA. The ability to form2-5A structures with double stranded RNA makes this system particularlyuseful for modulation of viral replication.

[0131] (2′-5′) oligoadenylate structures can be covalently linked toantisense molecules to form chimeric oligonucleotides capable of RNAcleavage (Torrence, supra). These molecules putatively bind and activatea 2-5A-dependent RNase, the oligonucleotide/enzyme complex then binds toa target RNA molecule which can then be cleaved by the RNase enzyme. Thecovalent attachment of 2′-5′ oligoadenylate structures is not limited toantisense applications, and can be further elaborated to includeattachment to nucleic acid molecules of the instant invention.

[0132] Enzymatic Nucleic Acid: Several varieties of naturally occurringenzymatic RNAs are presently known (Doherty and Doudna, 2001, Annu. Rev.Biophys. Biomol. Struct., 30, 457-475; Symons, 1994, Curr. Opin. Struct.Biol., 4, 322-30). In addition, several in vitro selection (evolution)strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have beenused to evolve new nucleic acid catalysts capable of catalyzing cleavageand ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87;Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, ScientificAmerican 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel etal., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumaret al., 1995, FASEB J, 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7,442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang etal., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long &Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al.,1997, Biochemistry 36, 6495). Each can catalyze a series of reactionsincluding the hydrolysis of phosphodiester bonds in trans (and thus cancleave other RNA molecules) under physiological conditions.

[0133] Nucleic acid molecules of this invention can block HBV proteinexpression and can be used to treat disease or diagnose diseaseassociated with the levels of HBV.

[0134] The enzymatic nature of an enzymatic nucleic acid has significantadvantages, such as the concentration of nucleic acid necessary toaffect a therapeutic treatment is low. This advantage reflects theability of the enzymatic nucleic acid molecule to act enzymatically.Thus, a single enzymatic nucleic acid molecule is able to cleave manymolecules of target RNA. In addition, the enzymatic nucleic acidmolecule is a highly specific modulator, with the specificity ofmodulation depending not only on the base-pairing mechanism of bindingto the target RNA, but also on the mechanism of target RNA cleavage.Single mismatches, or base-substitutions, near the site of cleavage canbe chosen to completely eliminate catalytic activity of an enzymaticnucleic acid molecule.

[0135] Nucleic acid molecules having an endonuclease enzymatic activityare able to repeatedly cleave other separate RNA molecules in anucleotide base sequence-specific manner. With proper design andconstruction, such enzymatic nucleic acid molecules can be targeted toany RNA transcript, and efficient cleavage achieved in vitro (Zaug etal., 324, Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al.,84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart.J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech,260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research1371, 1989; Chartrand et al., 1995, Nucleic Acids Research 23, 4092;Santoro et al., 1997, PNAS 94, 4262).

[0136] Because of their sequence specificity, trans-cleaving enzymaticnucleic acid molecules show promise as therapeutic agents for humandisease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294;Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymaticnucleic acid molecule can be designed to cleave specific RNA targetswithin the background of cellular RNA. Such a cleavage event renders theRNA non-functional and abrogates protein expression from that RNA. Inthis manner, synthesis of a protein associated with a disease state canbe selectively modulated (Warashina et al., 1999, Chemistry and Biology,6, 237-250.

[0137] The present invention also features nucleic acid sensor moleculesor allozymes having sensor domains comprising nucleic acid decoys and/oraptamers of the invention. Interaction of the nucleic acid sensormolecule's sensor domain with a molecular target, such as HBV RT and/orHBV RT primer, can activate or inactivate the enzymatic nucleic aciddomain of the nucleic acid sensor molecule, such that the activity ofthe nucleic acid sensor molecule is modulated in the presence of thetarget-signaling molecule. The nucleic acid sensor molecule can bedesigned to be active in the presence of the target molecule oralternately, can be designed to be inactive in the presence of themolecular target. For example, a nucleic acid sensor molecule isdesigned with a sensor domain having the sequence (UUCA)_(n), where n isan integer from 1-10. In a non-limiting example, interaction of the HBVRT primer with the sensor domain of the nucleic acid sensor molecule canactivate the enzymatic nucleic acid domain of the nucleic acid sensormolecule such that the sensor molecule catalyzes a reaction, for examplecleavage of HBV RNA. In this example, the nucleic acid sensor moleculeis activated in the presence of HBV RT or HBV RT primer, and can be usedas a therapeutic to treat HBV infection. Alternately, the reaction cancomprise cleavage or ligation of a labeled nucleic acid reportermolecule, providing a useful diagnostic reagent to detect the presenceof HBV in a system.

[0138] Synthesis of Nucleic acid Molecules

[0139] Synthesis of nucleic acids greater than 100 nucleotides in lengthis difficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., decoy nucleicacid molecules, aptamer nucleic acid molecules antisense nucleic acidmolecules, enzymatic nucleic acid molecules) are preferably used forexogenous delivery. The simple structure of these molecules increasesthe ability of the nucleic acid to invade targeted regions of proteinand/or RNA structure. Exemplary molecules of the instant invention arechemically synthesized, and others can similarly be synthesized.

[0140] Oligonucleotides (e.g., DNA oligonucleotides) are synthesizedusing protocols known in the art, for example as described in Carutherset al., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45sec coupling step for 2′-deoxy nucleotides. Table III outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be performedon a 96-well plate synthesizer, such as the instrument produced byProtogene (Palo Alto, Calif.) with minimal modification to the cycle. A33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramiditeand a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) canbe used in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol)of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxyresidues relative to polymer-bound 5′-hydroxyl. Average coupling yieldson the 394 Applied Biosystems, Inc. synthesizer, determined bycolorimetric quantitation of the trityl fractions, are typically97.5-99%. Other oligonucleotide synthesis reagents for the 394 AppliedBiosystems, Inc. synthesizer include the following: detritylationsolution is 3% TCA in methylene chloride (ABI); capping is performedwith 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10%2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mMpyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson SynthesisGrade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

[0141] Deprotection of the DNA-based oligonucleotides is performed asfollows: the polymer-bound trityl-on oligoribonucleotide is transferredto a 4 mL glass screw top vial and suspended in a solution of 40% aq.methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., thesupernatant is removed from the polymer support. The support is washedthree times with 1.0 mL of EtOH:MeCN:H20/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

[0142] The method of synthesis used for normal RNA including certaindecoy nucleic acid molecules and enzymatic nucleic acid moleculesfollows the procedure as described in Usman et al., 1987, J. Am. Chem.Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433;and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott etal., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end, and phosphoramidites at the 3′-end. In a non-limiting example,small scale syntheses are conducted on a 394 Applied Biosystems, Inc.synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling stepfor alkylsilyl protected nucleotides and a 2.5 min coupling step for2′-O-methylated nucleotides. Table III outlines the amounts and thecontact times of the reagents used in the synthesis cycle.Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-wellplate synthesizer, such as the instrument produced by Protogene (PaloAlto, Calif.) with minimal modification to the cycle. A 33-fold excess(60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-foldexcess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used ineach coupling cycle of 2′-O-methyl residues relative to polymer-bound5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl(ribo) protected phosphoramidite and a 150-fold excess of S-ethyltetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycleof ribo residues relative to polymer-bound 5′-hydroxyl. Average couplingyields on the 394 Applied Biosystems, Inc. synthesizer, determined bycolorimetric quantitation of the trityl fractions, are typically97.5-99%. Other oligonucleotide synthesis reagents for the 394 AppliedBiosystems, Inc. synthesizer include the following: detritylationsolution is 3% TCA in methylene chloride (ABI); capping is performedwith 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10%2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 49 mMpyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson SynthesisGrade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.

[0143] Deprotection of the RNA is performed using either a two-pot orone-pot protocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H20/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA•3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

[0144] Alternatively, for the one-pot protocol, the polymer-boundtrityl-on oligoribonucleotide is transferred to a 4 mL glass screw topvial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1(0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA•3HF (0.1mL) is added and the vial is heated at 65° C. for 15 min. The sample iscooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

[0145] For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 min. The cartridge is then washed again with water, salt exchangedwith 1 M NaCl and washed with water again. The oligonucleotide is theneluted with 30% acetonitrile.

[0146] Inactive hammerhead ribozymes or binding attenuated control (BAC)oligonucleotides are synthesized by substituting a U for G₅ and a U forA₁₄ (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20,3252). Similarly, one or more nucleotide substitutions can be introducedin other nucleic acid decoy molecules to inactivate the molecule andsuch molecules can serve as a negative control.

[0147] The average stepwise coupling yields are typically >98% (Wincottet al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skillin the art will recognize that the scale of synthesis can be adapted tobe larger or smaller than the example described above including but notlimited to 96-well format, all that is important is the ratio ofchemicals used in the reaction.

[0148] Alternatively, the nucleic acid molecules of the presentinvention can be synthesized separately and joined togetherpost-synthetically, for example, by ligation (Moore et al., 1992,Science 256, 9923; Draper et al., International PCT publication No. WO93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247;Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al.,1997, Bioconjugate Chem. 8, 204).

[0149] The nucleic acid molecules of the present invention can bemodified extensively to enhance stability by modification with nucleaseresistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro,2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17,34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes canbe purified by gel electrophoresis using general methods or can bepurified by high pressure liquid chromatography (HPLC; see Wincott etal., supra, the totality of which is hereby incorporated herein byreference) and re-suspended in water.

[0150] The sequences of the decoy constructs that are chemicallysynthesized, useful in this study, are shown in Table I. The decoyconstruct sequences listed in Table I can be formed of ribonucleotidesor other nucleotides or non-nucleotides. Such decoys are equivalent tothe decoy sequences described specifically in the Table.

[0151] Optimizing Activity of the Nucleic Acid Molecule of theInvention.

[0152] Chemically synthesizing nucleic acid molecules with modifications(base, sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

[0153] There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro,2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., International Publication PCT No. WO92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCTPublication No. WO 98/13526; Thompson et al., U.S. S No. 60/082,404which was filed on Apr. 20, 1998; Karpeisky et al., 1998, TetrahedronLett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic AcidSciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; allof the references are hereby incorporated in their totality by referenceherein). Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into ribozymes without modulating catalysis,and are incorporated by reference herein. In view of such teachings,similar modifications can be used as described herein to modify thenucleic acid molecules of the instant invention.

[0154] While chemical modification of oligonucleotide internucleotidelinkages with phosphorothioate, phosphorothioate, and/or5′-methylphosphonate linkages improves stability, excessivemodifications can cause some toxicity. Therefore, when designing nucleicacid molecules, the amount of these internucleotide linkages should beevaluated and minimized as necessary to lower toxicity, resulting inincreased efficacy and higher specificity of these molecules.

[0155] Nucleic acid molecules having chemical modifications thatmaintain or enhance activity are provided. Such a nucleic acid is alsogenerally more resistant to nucleases than an unmodified nucleic acid.Accordingly, the in vitro and/or in vivo activity should not besignificantly lowered. In cases in which modulation is the goal,therapeutic nucleic acid molecules delivered exogenously shouldoptimally be stable within cells until translation of the target RNA hasbeen modulated long enough to reduce the levels of the undesirableprotein. This period of time varies between hours to days depending uponthe disease state. Improvements in the chemical synthesis of RNA and DNA(Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al.,1992, Methods in Enzymology 211, 3-19 (incorporated by referenceherein)) have expanded the ability to modify nucleic acid molecules byintroducing nucleotide modifications to enhance their nucleasestability, as described above.

[0156] In one embodiment, nucleic acid molecules of the inventioninclude one or more G-clamp nucleotides. A G-clamp nucleotide is amodified cytosine analog wherein the modifications confer the ability tohydrogen bond both Watson-Crick and Hoogsteen faces of a complementaryguanine within a duplex, see for example Lin and Matteucci, 1998, J. Am.Chem. Soc., 120, 8531-8532. A single G-clamp analog substation within anoligonucleotide can result in substantially enhanced helical thermalstability and mismatch discrimination when hybridized to complementaryoligonucleotides. The inclusion of such nucleotides in nucleic acidmolecules of the invention results in both enhanced affinity andspecificity to nucleic acid targets. In another embodiment, nucleic acidmolecules of the invention include one or more LNA “locked nucleic acid”nucleotides, such as a 2′, 4′-C mythylene bicyclo nucleotide (see forexample Wengel et al., International PCT Publication No. WO 00/66604 andWO 99/14226).

[0157] In another embodiment, the invention features conjugates and/orcomplexes of nucleic acid molecules targeting HBV. Such conjugatesand/or complexes can be used to facilitate delivery of molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,phospholipids, nucleosides, nucleotides, nucleic acids, antibodies,toxins, negatively charged polymers and other polymers, for exampleproteins, peptides, hormones, carbohydrates, polyethylene glycols, orpolyamines, across cellular membranes. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system, with or without degradable linkers. Thesecompounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

[0158] The term “biodegradable nucleic acid linker molecule” as usedherein, refers to a nucleic acid molecule that is designed as abiodegradable linker to connect one molecule to another molecule, forexample, a biologically active molecule. The stability of thebiodegradable nucleic acid linker molecule can be modulated by usingvarious combinations of ribonucleotides, deoxyribonucleotides, andchemically modified nucleotides, for example, 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

[0159] The term “biodegradable” as used herein, refers to degradation ina biological system, for example enzymatic degradation or chemicaldegradation.

[0160] The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive molecules contemplated by the instant invention includetherapeutically active molecules such as antibodies, hormones,antivirals, peptides, proteins, chemotherapeutics, small molecules,vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

[0161] The term “phospholipid” as used herein, refers to a hydrophobicmolecule comprising at least one phosphorus group. For example, aphospholipid can comprise a phosphorus-containing group and saturated orunsaturated alkyl group, optionally substituted with OH, COOH, oxo,amine, or substituted or unsubstituted aryl groups.

[0162] Therapeutic nucleic acid molecules (e.g., decoy nucleic acidmolecules) delivered exogenously optimally are stable within cells untilreverse transcription of the pregenomic RNA has been modulated longenough to reduce the levels of HBV DNA. The nucleic acid molecules areresistant to nucleases in order to function as effective intracellulartherapeutic agents. Improvements in the chemical synthesis of nucleicacid molecules described in the instant invention and in the art haveexpanded the ability to modify nucleic acid molecules by introducingnucleotide modifications to enhance their nuclease stability asdescribed above.

[0163] In yet another embodiment, nucleic acid molecules having chemicalmodifications that maintain or enhance enzymatic activity are provided.Such nucleic acids are also generally more resistant to nucleases thanunmodified nucleic acids. Thus, in vitro and/or in vivo the activityshould not be significantly lowered. As exemplified herein, such nucleicacid molecules are useful in vitro and/or in vivo even if activity overall is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090).

[0164] Use of the nucleic acid-based molecules of the invention can leadto better treatment of the disease progression by affording thepossibility of combination therapies (e.g., multiple antisense, nucleicacid decoy, or nucleic acid aptamer molecules targeted to differentgenes; nucleic acid molecules coupled with known small moleculemodulators ors; or intermittent treatment with combinations of molecules(including different motifs) and/or other chemical or biologicalmolecules). The treatment of subjects with nucleic acid molecules canalso include combinations of different types of nucleic acid molecules.

[0165] In another aspect the nucleic acid molecules comprise a 5′ and/ora 3′-cap structure.

[0166] By “cap structure” is meant chemical modifications, which havebeen incorporated at either terminus of the oligonucleotide (see, forexample, Wincott et al., WO 97/26270, incorporated by reference herein).These terminal modifications protect the nucleic acid molecule fromexonuclease degradation, and can help in delivery and/or localizationwithin a cell. The cap can be present at the 5′-terminus (5′-cap) or atthe 3′-terminal (3′-cap) or can be present on both termini. Innon-limiting examples: the 5′-cap is selected from the group comprisinginverted abasic residue (moiety); 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details, seeWincott et al., International PCT publication No. WO 97/26270,incorporated by reference herein).

[0167] In another embodiment, the 3′-cap is selected from a groupcomprising, 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkylphosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropylphosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;alpha-nucleotide; modified base nucleotide; phosphorodithioate;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide;3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety;5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate;5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

[0168] By the term “non-nucleotide” is meant any group or compound whichcan be incorporated into a nucleic acid chain in the place of one ormore nucleotide units, including either sugar and/or phosphatesubstitutions, and allows the remaining bases to exhibit their enzymaticactivity. The group or compound is abasic in that it does not contain acommonly recognized nucleotide base, such as adenosine, guanine,cytosine, uracil or thymine.

[0169] An “alkyl” group refers to a saturated aliphatic hydrocarbon,including straight-chain, branched-chain, and cyclic alkyl groups. Inone embodiment, the alkyl group has 1 to 12 carbons, for example, it isa lower alkyl of from 1 to 7 carbons, more specifically 1 to 4 carbons.The alkyl group can be substituted or unsubstituted. When substitutedthe substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂ or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups thatare unsaturated hydrocarbon groups containing at least one carbon-carbondouble bond, including straight-chain, branched-chain, and cyclicgroups. In one embodiment, the alkenyl group has 1 to 12 carbons. Forexample, it can be a lower alkenyl of from 1 to 7 carbons, morespecifically 1 to 4 carbons. The alkenyl group can be substituted orunsubstituted. When substituted, the substituted group(s) can be,hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH.The term “alkyl” also includes alkynyl groups that have an unsaturatedhydrocarbon group containing at least one carbon-carbon triple bond,including straight-chain, branched-chain, and cyclic groups. In oneemobodiment, the alkynyl group has 1 to 12 carbons. For example, it canbe a lower alkynyl of from 1 to 7 carbons, more specifically 1 to 4carbons. The alkynyl group can be substituted or unsubstituted. Whensubstituted, the substituted group(s) can be, hydroxyl, cyano, alkoxy,═O, ═S, NO₂ or N(CH₃)₂, amino or SH.

[0170] Such alkyl groups can also include aryl, alkylaryl, carbocyclicaryl, heterocyclic aryl, amide and ester groups. An “aryl” group refersto an aromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′,where R is either alkyl, aryl, alkylaryl or hydrogen.

[0171] By “nucleotide” as used herein is as recognized in the art toinclude natural bases (standard), and modified bases well known in theart. Such bases are generally located at the 1′ position of a nucleotidesugar moiety. Nucleotides generally comprise a base, sugar and aphosphate group. The nucleotides can be unmodified or modified at thesugar, phosphate and/or base moiety, (also referred to interchangeablyas nucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents; such bases may be used at any position, for example, withinthe catalytic core of a nucleic acid decoy molecule and/or in thesubstrate-binding regions of the nucleic acid molecule.

[0172] In one embodiment, the invention features modified nucleic acids,for example decoys, with phosphate backbone modifications comprising oneor more phosphorothioate, phosphorodithioate, methylphosphonate,morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide,sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/oralkylsilyl, substitutions. For a review of oligonucleotide backbonemodifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues:Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, andMesmaeker et al., 1994, Novel Backbone Replacements forOligonucleotides, in Carbohydrate Modifications in Antisense Research,ACS, 24-39. These references are hereby incorporated by referenceherein.

[0173] By “abasic” is meant sugar moieties lacking a base or havingother chemical groups in place of a base at the 1′ position, (for moredetails, see Wincott et al., International PCT publication No. WO97/26270).

[0174] By “unmodified nucleoside” is meant one of the bases adenine,cytosine, guanine, thymine, uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

[0175] By “modified nucleoside” is meant any nucleotide base whichcontains a modification in the chemical structure of an unmodifiednucleotide base, sugar and/or phosphate.

[0176] In connection with 2′-modified nucleotides as described for thepresent invention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which may bemodified or unmodified. Such modified groups are described, for example,in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al.,WO 98/28317, which are both incorporated by reference in theirentireties.

[0177] Various modifications to nucleic acid (e.g., decoy, aptamer,antisense and ribozyme) structure can be made to enhance the utility ofthese molecules. Such modifications can enhance shelf-life, half-life invitro, stability, and ease of introduction of such oligonucleotides tothe target site, e.g., to enhance penetration of cellular membranes, andconfer the ability to recognize and bind to targeted cells.

[0178] Administration of Nucleic Acid Molecules Methods for the deliveryof nucleic acid molecules are described in Akhtar et al., 1992, TrendsCell Bio., 2, 139; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol.,16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137,165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all ofwhich are incorporated herein by reference. Sullivan et al., PCT WO94/02595, further describes the general methods for delivery ofenzymatic nucleic acid molecules. These protocols can be utilized forthe delivery of virtually any nucleic acid molecule. Nucleic acidmolecules can be administered to cells by a variety of methods known tothose of skill in the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres, or by proteinaceous vectors(O'Hare and Normand, International PCT Publication No. WO 00/53722).Alternatively, the nucleic acid/vehicle combination is locally deliveredby direct injection or by use of an infusion pump. Direct injection ofthe nucleic acid molecules of the invention, whether subcutaneous,intramuscular, or intradermal, can take place using standard needle andsyringe methodologies, or by needle-free technologies such as thosedescribed in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 andBarry et al., International PCT Publication No. WO 99/31262. Themolecules of the instant invention can be used as pharmaceutical agents.Pharmaceutical agents prevent, modulate the occurrence, ameliorate ortreat (alleviate a symptom to some extent, preferably all of thesymptoms) of a disease state in a subject.

[0179] Thus, the invention features a composition comprising one or morenucleic acid(s) of the invention in an pharmaceutically acceptablecarrier, such as a stabilizer, buffer, and the like. The negativelycharged polynucleotides of the invention can be administered (e.g., RNA,DNA or protein) and introduced into a subject by any standard means,with or without stabilizers, buffers, and the like, to form apharmaceutical composition. When it is desired to use a liposomedelivery mechanism, standard protocols for formation of liposomes can befollowed. The compositions of the present invention may also beformulated and used as tablets, capsules or elixirs for oraladministration, suppositories for rectal administration, sterilesolutions, suspensions for injectable administration, and the othercompositions known in the art.

[0180] The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

[0181] In one embodiment, a pharmaceutically acceptable composition orformulation refers to a composition or formulation in a form suitablefor administration, e.g., systemic administration, into a cell orsubject, including for example a human. Suitable forms, in part, dependupon the use or the route of entry, for example oral, transdermal, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell (i.e., a cell to which the negativelycharged nucleic acid is desirable for delivery). For example,compositions injected into the blood stream should be soluble. Otherfactors are known in the art, and include considerations such astoxicity and forms that prevent the composition or formulation fromexerting its effect.

[0182] By “systemic administration” is meant in vivo systemic absorptionor accumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes which lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes expose the desired negativelycharged polymers, e.g., nucleic acids, to an accessible diseased tissue.The rate of entry of a drug into the circulation has been shown to be afunction of molecular weight or size. The use of a liposome or otherdrug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach may provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells, such as cancer cells.

[0183] By “pharmaceutically acceptable formulation” is meant, acomposition or formulation that allows for the effective distribution ofthe nucleic acid molecules of the instant invention in the physicallocation most suitable for their desired activity. Nonlimiting examplesof agents suitable for formulation with the nucleic acid molecules ofthe instant invention include: P-glycoprotein inhibitors (such asPluronic P85), which can enhance entry of drugs into the CNS(Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13,16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide)microspheres for sustained release delivery after intracerebralimplantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58)(Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such asthose made of polybutylcyanoacrylate, which can deliver drugs across theblood brain barrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058.

[0184] The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

[0185] The present invention also includes compositions prepared forstorage or administration, which include a pharmaceutically effectiveamount of the desired compounds in a pharmaceutically acceptable carrieror diluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985) hereby incorporated by reference herein. For example,preservatives, stabilizers, dyes and flavoring agents may be provided.These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentsmay be used.

[0186] A pharmaceutically effective amount or dose is that dose requiredto prevent, inhibit the occurrence of, ameliorate or treat (alleviate asymptom to some extent, preferably all of the symptoms) a disease state.The pharmaceutically effective dose depends on the type of disease, thecomposition used, the route of administration, the type of mammal beingtreated, the physical characteristics of the specific mammal underconsideration, concurrent medication, and other factors that thoseskilled in the medical arts will recognize. Generally, an amount between0.1 mg/kg and 100 mg/kg body weight/day of active ingredients isadministered dependent upon potency of the negatively charged polymer.

[0187] The present invention also includes compositions prepared forstorage or administration that include a pharmaceutically effectiveamount of the desired compounds in a pharmaceutically acceptable carrieror diluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985), hereby incorporated by reference herein. For example,preservatives, stabilizers, dyes and flavoring agents can be provided.These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentscan be used.

[0188] The nucleic acid molecules of the invention and formulationsthereof can be administered orally, topically, parenterally, byinhalation or spray, or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand/or vehicles. The term parenteral as used herein includespercutaneous, subcutaneous, intravascular (e.g., intravenous),intramuscular, or intrathecal injection or infusion techniques and thelike. In addition, there is provided a pharmaceutical formulationcomprising a nucleic acid molecule of the invention and apharmaceutically acceptable carrier. One or more nucleic acid moleculesof the invention can be present in association with one or morenon-toxic pharmaceutically acceptable carriers and/or diluents and/oradjuvants, and if desired other active ingredients. The pharmaceuticalcompositions containing nucleic acid molecules of the invention can bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsion, hard or soft capsules, or syrups or elixirs.

[0189] Compositions intended for oral use can be prepared according toany method known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

[0190] Formulations for oral use can also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin or oliveoil.

[0191] Aqueous suspensions contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, for example, sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

[0192] Oily suspensions can be formulated by suspending the activeingredients in a vegetable oil, for example arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.The oily suspensions can contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoringagents can be added to provide palatable oral preparations. Thesecompositions can be preserved by the addition of an anti-oxidant such asascorbic acid.

[0193] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water provide the activeingredient in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents or suspending agents are exemplified by those already mentionedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, can also be present.

[0194] Pharmaceutical compositions of the invention can also be in theform of oil-in-water emulsions. The oily phase can be a vegetable oil ora mineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

[0195] Syrups and elixirs can be formulated with sweetening agents, forexample glycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono-or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

[0196] The nucleic acid molecules of the invention can also beadministered in the form of suppositories, e.g., for rectaladministration of the drug. These compositions can be prepared by mixingthe drug with a suitable non-irritating excipient that is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter and polyethylene glycols.

[0197] Nucleic acid molecules of the invention can be administeredparenterally in a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

[0198] Dosage levels of the order of from about 0.1 mg to about 140 mgper kilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

[0199] It is understood that the specific dose level for any particularsubject depends upon a variety of factors including the activity of thespecific compound employed, the age, body weight, general health, sex,diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy.

[0200] For administration to non-human animals, the composition can alsobe added to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

[0201] The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication may increase the beneficialeffects while reducing the presence of side effects.

[0202] In one embodiment, the invention compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes, such as hepatocytes. For example, the asialoglycoprotein receptor(ASGPr) (Wu and Wu, 1987, J Biol. Chem. 262, 4429-4432) is unique tohepatocytes and binds branched galactose-terminal glycoproteins, such asasialoorosomucoid (ASOR). Binding of such glycoproteins or syntheticglycoconjugates to the receptor takes place with an affinity thatstrongly depends on the degree of branching of the oligosaccharidechain, for example, triatennary structures are bound with greateraffinity than biatenarry or monoatennary chains (Baenziger and Fiete,1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257,939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtainedthis high specificity through the use of N-acetyl-D-galactosamine as thecarbohydrate moiety, which has higher affinity for the receptor,compared to galactose. This “clustering effect” has also been describedfor the binding and uptake of mannosyl-terminating glycoproteins orglycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395).The use of galactose and galactosamine based conjugates to transportexogenous compounds across cell membranes can provide a targeteddelivery approach to the treatment of liver disease such as HBVinfection or hepatocellular carcinoma. The use of bioconjugates can alsoprovide a reduction in the required dose of therapeutic compoundsrequired for treatment. Furthermore, therapeutic bioavialability,pharmacodynamics, and pharmacokinetic parameters can be modulatedthrough the use of nucleic acid bioconjugates of the invention.

[0203] Alternatively, certain of the nucleic acid molecules of theinstant invention can be expressed within cells from eukaryoticpromoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarryand Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon etal., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet etal., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J.Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4;Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen etal., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science,247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259;Good et al., 1997, Gene Therapy, 4, 45; all of these references arehereby incorporated in their totalities by reference herein). Thoseskilled in the art realize that any nucleic acid can be expressed ineukaryotic cells from the appropriate DNA/RNA vector. The activity ofsuch nucleic acids can be augmented by their release from the primarytranscript by a ribozyme (Draper et al., PCT WO 93/23569, and Sullivanet al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser.,27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Venturaet al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, JBiol. Chem., 269, 25856; all of these references are hereby incorporatedin their totality by reference herein).

[0204] In another aspect of the invention, nucleic acid molecules of thepresent invention are preferably expressed from transcription units(see, for example, Couture et al., 1996, TIG., 12, 510) inserted intoDNA or RNA vectors. The recombinant vectors are preferably DNA plasmidsor viral vectors. Nucleic acid expressing viral vectors could beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. Preferably, the recombinantvectors capable of expressing the nucleic acid molecules are deliveredas described above, and persist in target cells. Alternatively, viralvectors can be used that provide for transient expression of nucleicacid molecules. Such vectors can be repeatedly administered asnecessary. Once expressed, the nucleic acid molecule binds to the targetmRNA. Delivery of nucleic acid molecule expressing vectors can besystemic, such as by intravenous or intra-muscular administration, byadministration to target cells ex-planted from the subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell (for a review see Coutureet al., 1996, TIG., 12, 510).

[0205] In one aspect, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one of the nucleicacid molecules of the instant invention. The nucleic acid sequenceencoding the nucleic acid molecule of the instant invention is operablylinked in a manner which allows expression of that nucleic acidmolecule.

[0206] In another aspect the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); c) a nucleicacid sequence encoding at least one of the nucleic acid catalyst of theinstant invention; and wherein said sequence is operably linked to saidinitiation region and said termination region, in a manner which allowsexpression and/or delivery of said nucleic acid molecule. The vector canoptionally include an open reading frame (ORF) for a protein operablylinked on the 5′ side or the 3′-side of the sequence encoding thenucleic acid catalyst of the invention; and/or an intron (interveningsequences).

[0207] Transcription of the nucleic acid molecule sequences are drivenfrom a promoter for eukaryotic RNA polymerase I (pol I), RNA polymeraseII (pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onthe nature of the gene regulatory sequences (enhancers, silencers, etc.)present nearby. Prokaryotic RNA polymerase promoters are also used,providing that the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci.USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72;Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990,Mol. Cell. Biol., 10, 4529-37). All of these references are incorporatedby reference herein. Several investigators have demonstrated thatnucleic acid molecules, such as ribozymes expressed from such promoterscan function in mammalian cells (e.g. Kashani-Sabet et al., 1992,Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4;L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993,Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4; Thompson et al., 1995, NucleicAcids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). Morespecifically, transcription units such as the ones derived from genesencoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VARNA are useful in generating high concentrations of desired RNAmolecules such as ribozymes in cells (Thompson et al., supra; Coutureand Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res.,22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997,Gene Ther., 4, 45; Beigelman et al., International PCT Publication No.WO 96/18736; all of these publications are incorporated by referenceherein). The above ribozyme transcription units can be incorporated intoa variety of vectors for introduction into mammalian cells, includingbut not restricted to, plasmid DNA vectors, viral DNA vectors (such asadenovirus or adeno-associated virus vectors), or viral RNA vectors(such as retroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

[0208] In yet another aspect, the invention features an expressionvector comprising nucleic acid sequence encoding at least one of thenucleic acid molecules of the invention, in a manner that allowsexpression of that nucleic acid molecule. The expression vectorcomprises in one embodiment; a) a transcription initiation region; b) atranscription termination region; c) a nucleic acid sequence encoding atleast one said nucleic acid molecule; and wherein said sequence isoperably linked to said initiation region and said termination region,in a manner which allows expression and/or delivery of said nucleic acidmolecule. In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; d) a nucleic acid sequence encoding at leastone said nucleic acid molecule, wherein said sequence is operably linkedto the 3′-end of said open reading frame; and wherein said sequence isoperably linked to said initiation region, said open reading frame andsaid termination region, in a manner which allows expression and/ordelivery of said nucleic acid molecule. In yet another embodiment, theexpression vector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; d) a nucleic acidsequence encoding at least one said nucleic acid molecule; and whereinsaid sequence is operably linked to said initiation region, said intronand said termination region, in a manner which allows expression and/ordelivery of said nucleic acid molecule. In another embodiment, theexpression vector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; d) an open readingframe; e) a nucleic acid sequence encoding at least one said nucleicacid molecule, wherein said sequence is operably linked to the 3′-end ofsaid open reading frame; and wherein said sequence is operably linked tosaid initiation region, said intron, said open reading frame and saidtermination region, in a manner which allows expression and/or deliveryof said nucleic acid molecule.

EXAMPLES

[0209] The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

[0210] The following examples demonstrate the selection and design ofnucleic acid decoy molecules that target HBV reverse transcriptase.

Example 1 Modulation of HBV Reverse Transcriptase

[0211] The HBV reverse transcriptase (pol) binds to the 5′ stem-loopstructure in the HBV pregenomic RNA and synthesizes a four-nucleotideprimer from the template UUCA. The reverse transcriptase thentranslocates to the 3′ end of the pregenomic RNA where the primer bindsto the UUCA sequence within the DR1 element and begins first-strandsynthesis of HBV DNA. A number of short oligos, ranging in size from 4to 16-mers, were designed to act as competitive inhibitors of the HBVreverse transcriptase primer, either by blocking the primer bindingsites on the HBV RNA or by acting as a decoy.

[0212] The oligonucleotides and controls were synthesized in all2′-O-methyl and 2′-O-allyl versions (Table II). The inverse sequence ofall oligos were generated to serve as controls. Primary screening of thecompetitive inhibitors was completed in the HBsAg transfection/ELISAsystem, in which the oligo is co-transfeceted with a HBV cDNA vectorinto Hep G2 cells. Following 4 days of incubation, the levels of HBsAgsecreted into the cell culture media were determined by ELISA. Screeningof the 2′-O-allyl versions revealed that two of the decoy oligos(RPI.24944 and RPI.24945), consisting of 3× or 4×repeats of the RTprimer binding site UUCA, along with the matched inverse controls,displayed considerable activity by decreasing HBsAg levels (FIG. 3).This dramatic decrease in HBsAg levels is not due to cellular toxicity,because a MTS assay showed no difference in proliferation between any ofthe treated cells. A follow up experiment with a 5×UUCA repeat, theinverse sequence control, and a matched scrambled control, showed thatall three oligos decreased HBsAg levels without cellular toxicity.Screening of the 2′-O-methyl versions of the oligos showed no activityfrom the 3× and 4×UUCA repeat (FIG. 4), suggesting that the anti-HBVeffect could be related to the 2′-O-allyl chemistry rather than tosequence specificity.

[0213] Screening of the 2′-O-methyl oligos did show that the 2′-O-methyl2×UUCA repeat, RPI.24986, displayed activity in decreasing HBsAg levelsas compared to the inverse control, RPI.24950. A dose responseexperiment showed that at the lower concentrations of 100 and 200 nM,RPI.24986 showed greater activity in decreasing HbsAg levels as comparedto the inverse control RPI.24950 (FIG. 5).

Example 2 Modulation of HBV Transcription via Oligonucleotides Targetingthe Enchancer I Core Region of HBV DNA

[0214] In an effort to block HBV replication, oligonucleotides weredesigned to bind to two liver-specific factor binding sites in theEnhancer I core region of HBV genomic DNA. Hepatocyte Nuclear Factor 3(HNF3) and Hepatocyte Nuclear Factor 4 (HNF4) bind to sites in the coreregion, with the HNF3 site being 5′ to the HNF4 site. The HNF3 and HNF4sites overlap or are adjacent to binding sites for a number of moreubiquitous factors, and are termed nuclear receptor response elements(NRRE). These elements are critical in regulating HBV transcription andreplication in infected hepatocytes, with mutations in the HNF3 and HNF4binding sites having been demonstrated to greatly reduce the levels ofHBV replication (Bock et al., 2000, J. Virology, 74, 2193)

[0215] Oligonucleotides (Table II) were designed to bind to either thepositive or negative strands of the HNF3 or HNF4 binding sites.Scrambled controls were made to match each oligo. Each oligo wassynthesized in all 2′-O-methyl/all phosphorothioate, or all2′-O-allyl/all phosphorothioate chemistries. The initial screening ofthe oligos was done in the HBsAg transfection/ELISA system in Hep G2cells as described in Example 1. RPI.25654, which targets the negativestrand of the HNF4 binding site, shows greater activity in reducingHBsAg levels as compared to RPI.25655, which targets the HNF4 sitepositive strand, and the scrambled control RPI.25656. This result wasobserved at both 200 and 400 nM (FIGS. 6 and 7). In a follow-up study,RPI.25654 reduced HBsAg levels in a dose-dependent manner, from 50-200nM (FIG. 8).

Example 3 Transfection of HepG2 Cells With psHBV-1 and Nucleic Acid

[0216] The human hepatocellular carcinoma cell line Hep G2 was grown inDulbecco's modified Eagle media supplemented with 10% fetal calf serum,2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,25 mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. Togenerate a replication competent cDNA, prior to transfection the HBVgenomic sequences are excised from the bacterial plasmid sequencecontained in the psHBV-1 vector This was done with an EcoRI and Hind IIIrestriction digest. Following completion of the digest, a ligation wasperformed under dilute conditions (20 μg/ml) to favor intermolecularligation. The total ligation mixture was then concentrated using Qiagenspin columns. One skilled in the art would realize that other methodscan be used to generate a replication competent cDNA

[0217] Secreted alkaline phosphatase (SEAP) was used to normalize theHBsAg levels to control for transfection variability. The pSEAP2-TKcontrol vector was constructed by ligating a Bgl II-Hind III fragment ofthe pRL-TK vector (Promega), containing the herpes simplex virusthymidine kinase promoter region, into Bgl II/Hind III digestedpSEAP2-Basic (Clontech). Hep G2 cells were plated (3×10⁴ cells/well) in96-well microtiter plates and incubated overnight. A lipid/DNA/nucleicacid complex was formed containing (at final concentrations) cationiclipid (15 μg/ml), prepared psHBV-1 (4.5 μg/ml), pSEAP2-TK (0.5 μg/ml),and nucleic acid (100 μM). Following a 15 min. incubation at 37° C., thecomplexes were added to the plated Hep G2 cells. Media was removed fromthe cells 96 hour post-transfection for HBsAg and SEAP analysis.

[0218] Transfection of the human hepatocellular carcinoma cell line, HepG2, with replication competent HBV DNA results in the expression of HBVproteins and the production of virions.

Example 4 Analysis of HBsAg and SEAP Levels Following Nucleic AcidTreatment

[0219] Immulon 4 (Dynax) microtiter wells were coated overnight at 4° C.with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1 μg/ml in CarbonateBuffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5). The wells were then washed4× with PBST (PBS, 0.05% Tween® 20) and blocked for 1 hr at 37° C. withPBST, 1% BSA. Following washing as above, the wells were dried at 37° C.for 30 minutes. Biotinylated goat anti-HBsAg (Accurate YVS 1807) wasdiluted 1:1000 in PBST and incubated in the wells for 1 hour at 37° C.The wells were washed 4× with PBST. Streptavidin/Alkaline PhosphataseConjugate (Pierce 21324) was diluted to 250 ng/ml in PBST, and incubatedin the wells for 1 hour at 37° C. After washing as above, p-nitrophenylphosphate substrate (Pierce 37620) was added to the wells, which werethen incubated for 1 hour at 37° C. The optical density at 405 nm wasthen determined. SEAP levels were assayed using the Great EscAPe®Detection Kit (Clontech K2041-1), as per the manufacturers instructions.

Example 5 Analysis of HBV DNA Expression a HepG2.2.15 Murine Model

[0220] The development of new antiviral agents for the treatment ofchronic Hepatitis B has been aided by the use of animal models that arepermissive to replication of related Hepadnaviridae such as WoodchuckHepatitis Virus (WHV) and Duck Hepatitis Virus (DHV). In addition, theuse of transgenic mice has also been employed. The human hepatoblastomacell line, HepG2.2.15, implanted as a subcutaneous (SC) tumor, can beused to produce Hepatitis B viremia in mice. This model is useful forevaluating new HBV therapies. Mice bearing HepG2.2.15 SC tumors show HBVviremia. HBV DNA can be detected in serum beginning on Day 35. Maximumserum viral levels reach 1.9×10⁵ copies/mL by day 49. A study alsodetermined that the minimum tumor volume associated with viremia was 300mm³. Therefore, the HepG2.2.15 cell line grown as a SC tumor produces auseful model of HBV viremia in mice. This new model can be used toevaluate new therapeutic regimens for chronic Hepatitis B.

[0221] HepG2.2.15 tumor cells contain a slightly truncated version ofviral HBV DNA and sheds HBV particles. The purpose of this study was toidentify what time period viral particles are shed from the tumor. Serumwas analyzed for presence of HBV DNA over a time course after HepG2.2.15tumor inoculation in Athymic Ncr nu/nu mice. HepG2.2.15 cells werecarried and expanded in DMEM/10% FBS/2.4% HEPES/1% NEAA/1% Glutamine/1%Sodium Pyruvate media. Cells were resuspended in Delbecco's PBS withcalcium/magnesium for injection. One hundred microliters of the tumorcell suspension (at a concentration of 1×10⁸ cells/mL) were injectedsubcutaneously in the flank of NCR nu/nu female mice with a 23 μl needleand 1 cc syringe, thereby giving each mouse 1×10⁷ cells. Tumors wereallowed to grow for a period of up to 49 days post tumor cellinoculation. Serum was sampled for analysis on days 1, 7, 14, 35, 42 and49 post tumor inoculation. Length and width measurements from each tumorwere obtained three times per week using a Jamison microcaliper. Tumorvolumes were calculated from tumor length/width measurements (tumorvolume=0.5[a(b)²] where a=longest axis of the tumor and b=shortest axisof the tumor). Serum was analyzed for the presence of HBV DNA by theRoche Amplicor HBV moniter TM DNA assay.

[0222] Cell Culture Models

[0223] As previously mentioned, HBV does not infect cells in culture.However, transfection of HBV DNA (either as a head-to-tail dimer or asan “overlength” genome of >100%) into HuH7 or Hep G2 hepatocytes resultsin viral gene expression and production of HBV virions released into themedia. Thus, HBV replication competent DNA are co-transfected withribozymes in cell culture. Such an approach has been used to reportintracellular ribozyme activity against HBV (zu Putlitz, et al., 1999,J. Virol., 73, 5381-5387, and Kim et al., 1999, Biochem. Biophys. Res.Commun., 257, 759-765). In addition, stable hepatocyte cell lines havebeen generated that express HBV. In these cells, only ribozyme need bedelivered; however, performance of a delivery screen is required.Intracellular HBV gene expression can be assayed by a Taqman® assay forHBV RNA or by ELISA for HBV protein. Extracellular virus can be assayedby PCR for DNA or ELISA for protein. Antibodies are commerciallyavailable for HBV surface antigen and core protein. A secreted alkalinephosphatase expression plasmid can be used to normalize for differencesin transfection efficiency and sample recovery.

[0224] Animal Models

[0225] There are several small animal models to study HBV replication.One is the transplantation of HBV-infected liver tissue into irradiatedmice. Viremia (as evidenced by measuring HBV DNA by PCR) is firstdetected 8 days after transplantation and peaks between 18-25 days (Ilanet al., 1999, Hepatology, 29, 553-562).

[0226] Transgenic mice that express HBV have also been used as a modelto evaluate potential anti-virals. HBV DNA is detectable in both liverand serum (Guidotti et al., 1995, J. Virology, 69, 10, 6158-6169; Morreyet al., 1999, Antiviral Res., 42, 97-108).

[0227] An additional model is to establish subcutaneous tumors in nudemice with Hep G2 cells transfected with HBV. Tumors develop in about 2weeks after inoculation and express HBV surface and core antigens. HBVDNA and surface antigen is also detected in the circulation oftumor-bearing mice (Yao et al., 1996, J. Viral Hepat., 3, 19-22).

[0228] In one embodiment, the invention features a mouse, for example amale or female mouse, implanted with HepG2.2.15 cells, wherein the mouseis susceptible to HBV infection and capable of sustaining HBV DNAexpression. One embodiment of the invention provides a mouse implantedwith HepG2.2.15 cells, wherein said mouse sustains the propagation ofHEPG2.2.15 cells and HBV production (see Macejak, U.S. ProvisionalPatent Application No. 60/296,876).

[0229] Woodchuck hepatitis virus (WHV) is closely related to HBV in itsvirus structure, genetic organization, and mechanism of replication. Aswith HBV in humans, persistent WHV infection is common in naturalwoodchuck populations and is associated with chronic hepatitis andhepatocellular carcinoma (HCC). Experimental studies have establishedthat WHV causes HCC in woodchucks and woodchucks chronically infectedwith WHV have been used as a model to lest a number of anti-viralagents. For example, the nucleoside analogue 3T3 was observed to causedose dependent reduction in virus (50% reduction after two dailytreatments at the highest dose) (Hurwitz et al., 1998. Antimicrob.Agents Chemother., 42, 2804-2809).

[0230] Indications

[0231] Particular degenerative and disease states that can be associatedwith HBV expression modulation include, but are not limited to, HBVinfection, hepatitis, cancer, tumorigenesis, cirrhosis, liver failureand others.

[0232] The present body of knowledge in HBV research indicates the needfor methods to assay HBV activity and for compounds that can regulateHBV expression for research, diagnostic, and therapeutic use.

[0233] Lamivudine (3TC®), L-FMAU, adefovir dipivoxil, type 1 Interferon(e.g, interferon alpha, interferon beta, consensus interferon,polyethylene glycol interferon, polyethylene glycol interferon alpha 2a,polyethylene glycol interferon 2b, and polyethylene glycol consensusinterferon), therapeutic vaccines, steriods, and 2′-5′ Oligoadenylatesare non-limiting examples of pharmaceutical agents that can be combinedwith or used in conjunction with the nucleic acid molecules of theinstant invention. Those skilled in the art will recognize that otherdrugs or other therapies can similarly and readily be combined with thenucleic acid molecules of the instant invention and are, therefore,within the scope of the instant invention.

[0234] All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

[0235] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

[0236] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention. Thus, such additional embodiments are within the scope of thepresent invention and the following claims.

[0237] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments, optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by thedescription and the appended claims.

[0238] In addition, where features or aspects of the invention aredescribed in terms of Markush groups or other grouping of alternatives,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group or other group. TABLE I HBV RT primer Decoysequences Seq ID Length Decoy Sequence No. 4 AUUC 1 4 CAUU 2 4 UCAU 3 4UUCA 4 5 AUUCA 5 5 CAUUC 6 5 UCAUU 7 5 UUCAU 8 6 AUUCAU 9 6 CAUUCA 10 6UCAUUC 11 6 UUCAUU 12 7 AUUCAUU 13 7 CAUUCAU 14 7 UCAUUCA 15 7 UUCAUUC16 8 AUUCAUUC 17 8 CAUUCAUU 18 8 UCAUUCAU 19 8 UUCAUUCA 20 9 AUUCAUUCA21 9 CAUUCAUUC 22 9 UCAUUCAUU 23 9 UUCAUUCAU 24 10 AUUCAUUCAU 25 10CAUUCAUUCA 26 10 UCAUUCAUUC 27 10 UUCAUUCAUU 28 11 AUUCAUUCAUU 29 11CAUUCAUUCAU 30 11 UCAUUCAUUCA 31 11 UUCAUUCAUUC 32 12 AUUCAUUCAUUC 33 12CAUUCAUUCAUU 34 12 UCAUUCAUUCAU 35 12 UUCAUUCAUUCA 36 13 AUUCAUUCAUUCA37 13 CAUUCAUUCAUUC 38 13 UCAUUCAUUCAUU 39 13 UUCAUUCAUUCAU 40 14AUUCAUUCAUUCAU 41 14 CAUUCAUUCAUUCA 42 14 UCAUUCAUUCAUUC 43 14UUCAUUCAUUCAUU 44 15 AUUCAUUCAUUCAUU 45 15 CAUUCAUUCAUUCAU 46 15UCAUUCAUUCAUUCA 47 15 UUCAUUCAUUCAUUC 48 16 AUUCAUUCAUUCAUUC 49 16CAUUCAUUCAUUCAUU 50 16 UCAUUCAUUCAUUCAU 52 16 UUCAUUCAUUCAUUCA 52 17AUUCAUUCAUUCAUUCA 53 17 CAUUCAUUCAUUCAUUC 54 17 UCAUUCAUUCAUUCAUU 55 17UUCAUUCAUUCAUUCAU 56 18 AUUCAUUCAUUCAUUCAU 57 18 CAUUCAUUCAUUCAUUCA 5818 UCAUUCAUUCAUUCAUUC 59 18 UUCAUUCAUUCAUUCAUU 60 19 AUUCAUUCAUUCAUUCAUU61 19 CAUUCAUUCAUUCAUUCAU 62 19 UCAUUCAUUCAUUCAUUCA 63 19UUCAUUCAUUCAUUCAUUC 64 20 AUUCAUUCAUUCAUUCAUUC 65 20CAUUCAUUCAUUCAUUCAUU 66 20 UCAUUCAUUCAUUCAUUCAU 67 20UUCAUUCAUUCAUUCAUUCA 68 21 AUUCAUUCAUUCAUUCAUUCA 69 21CAUUCAUUCAUUCAUUCAUUC 70 21 UCAUUCAUUCAUUCAUUCAUU 71 21UUCAUUCAUUCAUUCAUUCAU 72 22 CAUUCAUUCAUUCAUUCAUUCA 73 22UCAUUCAUUCAUUCAUUCAUUC 74 22 UUCAUUCAUUCAUUCAUUCAUU 75 23UCAUUCAUUCAUUCAUUCAUUCA 76 23 UUCAUUCAUUCAUUCAUUCAUUC 77 24UUCAUUCAUUCAUUCAUUCAUUCA 78

[0239] TABLE II Synthetic Nucleic acid molecules RPI# Alias SequenceSeqID 24961 HBV DR1 2′Oallyl P = S g_(s)c_(s)a_(s)g_(s)a_(s)g_(s)g_(s)u_(s)g_(s)a_(s)a_(s)B 79 24997 HBV DR12′Oallyl P = S control a_(s)a_(s)g_(s)u_(s)g_(s)g_(s)a_(s)g_(s)a_(s)c_(s)g_(s)B 80 24956 HBV1866-1869 1 × 2′Oallyl P = S u _(s)u_(s)c_(s)a_(s)B 81 24992 HBV1866-1869 1 × 2′Oallyl P = S control a _(s)c_(s)u_(s)u_(s)B 82 24941 HBV1866-1869 2 × 2′Oallyl P = S u _(s)u_(s)c_(s)a_(s)u_(s)u_(s)c_(s)a_(s)B83 24959 HBV 1866-1869 2 × 2′Oallyl P = S control a_(s)c_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)B 84 24944 HBV 1866-1869 3× 2′Oallyl P =S u_(s)u_(s)c_(s)a_(s)u_(s)u_(s)c_(s)a_(s)u_(s)u_(s)c_(s)a_(s)B 85 24962HBV 1866-1869 3 × 2′Oallyl P = S control a_(s)c_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)B 86 24945HBV 1866-1869 4 × 2′Oallyl P = S u_(s)u_(s)c_(s)a_(s)u_(s)u_(s)c_(s)a_(s)u_(s)u_(s)c_(s)a_(s)u_(s)u_(s)c_(s)a_(s)B87 24963 HBV 1866-1869 4 × 2′Oallyl P = S control a_(s)c_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)B88 24938 HBV 1866-1869 2′Oallyl P = S u _(s)g_(s)a_(s)a_(s)B 89 24974HBV 1866-1869 2′Oallyl P = S control a _(s)a_(s)g_(s)u_(s)B 90 24940 HBV1866-1872 2′Oallyl P = S g _(s)c_(s)u_(s)u_(s)g_(s)a_(s)a_(s)B 91 24958HBV 1866-1872 2′Oallyl P = S control a_(s)a_(s)g_(s)u_(s)u_(s)c_(s)g_(s)B 92 24943 HBV 1866-1876 2′Oallyl P= S g _(s)g_(s)a_(s)g_(s)g_(s)c_(s)u_(s)u_(s)g_(s)a_(s)aB 93 24979 HBV1866-1876 2′Oallyl P = S control a_(s)a_(s)g_(s)u_(s)u_(s)c_(s)g_(s)g_(s)a_(s)g_(s)g_(s)B 94 18341 HBV-273UH.Rz-7 allyl stab1 g_(s)a_(s)a_(s)a_(s)auu cUGAuGaggccguuaggccGaa 95Agagaag B 24588 HBV-273 UH.Rz-7 allyl stab1 inact3 scram1a_(s)a_(s)u_(s)g_(s)agg cUAGuGacgccguuaggcgGaa 96 (GUUA SAC) Aaaugaa B24929 HBV 1866-1969 2′Omethyl ugaaB 97 24965 HBV 1866-1969 2′Omethylcontrol aaguB 98 24934 HBV 1866-1876 2′Omethyl ggaggcuugaaB 99 24970 HBV1866-1876 2′Omethyl control aaguucggaggB 100 24976 HBV 1866-18722′Omethyl gcuugaaB 101 24949 HBV 1866-1872 2′Omethyl control aaguucgB102 24952 HBV DR1 2′Omethyl gcagaggugaaB 103 24988 HBV DR1 2′Omethylcontrol aaguggagacgB 104 24947 HBV 1866-1869 1 × 2′Omethyl uucaB 10524983 HBV 1866-1869 1 × 2′Omethyl control acuuB 106 24986 HBV 1866-18692 × 2′Omethyl uucauucaB 107 24950 HBV 1866-1869 2 × 2′Omethyl controlacuuacuuB 108 24989 HBV 1866-1869 3 × 2′Omethyl uucauucauucaB 109 24953HBV 1866-1869 3 × 2′Omethyl control acuuacuuacuuB 110 24936 HBV1866-1869 4 × 2′Omethyl uucauucauucauucaB 111 24954 HBV 1866-1869 4× 2′Omethyl control acuuacuuacuuacuuB 112 25639 HBV 5′ Enl pos OMe P = SBu_(s)u_(s)u_(s)c_(s)u_(s)a_(s)a_(s)g_(s)u_(s)a_(s)a_(s)a_(s)c_(s)a_(s)g_(s)uB 113 25640 HBV 5′ Enl neg OMe P = S Ba_(s)c_(s)u_(s)g_(s)u_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)a_(s)g_(s)a_(s)a_(s)aB 114 25641 HBV 5′ Enl sc OMe P = S Ba_(s)a_(s)g_(s)u_(s)a_(s)a_(s)c_(s)u_(s)c_(s)u_(s)a_(s)u_(s)g_(s)u_(s)u_(s)aB 115 25642 HBV 3′ Enl pos OMe P = S Bu_(s)a_(s)c_(s)a_(s)u_(s)g_(s)a_(s)a_(s)c_(s)c_(s)u_(s)u_(s)u_(s)a_(s)c_(s)c_(s)c_(s)cB 116 25643 HBV 3′ Enl neg OMe P = S Bg_(s)g_(s)g_(s)u_(s)a_(s)a_(s)a_(s)g_(s)g_(s)u_(s)u_(s)c_(s)a_(s)u_(s)g_(s)u_(s)aB 117 25644 HBV 3′ Enl pos sc OMe P = S Ba_(s)c_(s)c_(s)u_(s)a_(s)u_(s)c_(s)g_(s)c_(s)c_(s)u_(s)a_(s)c_(s)u_(s)c_(s)u_(s)a_(s)aB 118 25645 HBV 5′ Enl neg sc OMe P = S Bu_(s)g_(s)a_(s)u_(s)a_(s)g_(s)c_(s)g_(s)g_(s)a_(s)u_(s)g_(s)a_(s)g_(s)a_(s)u_(s)uB 119 25646 HBV DR1 pos OMe P = S Bu_(s)u_(s)c_(s)a_(s)c_(s)c_(s)u_(s)c_(s)u_(s)g_(s)c B 120 25651 HBV5′ Enl pos Oallyl P = S B u_(s)u_(s)u_(s)c_(s)u_(s)a_(s)a_(s)g_(s)u_(s)a_(s)a_(s)a_(s)c_(s)a_(s)g_(s)uB 121 25652 HBV 5′ Enl neg Oallyl P = S B a_(s)c_(s)u_(s)g_(s)u_(s)u_(s)u_(s)a_(s)c_(s)u_(s)u_(s)a_(s)g_(s)a_(s)a_(s)aB 122 25653 HBV 5′ Enl sc Oallyl P = S B a_(s)a_(s)g_(s)u_(s)a_(s)a_(s)c_(s)u_(s)c_(s)u_(s)a_(s)u_(s)g_(s)u_(s)u_(s)aB 123 25654 HBV 3′ Enl pos Oallyl P = S B u_(s)a_(s)c_(s)a_(s)u_(s)g_(s)a_(s)a_(s)c_(s)c_(s)u_(s)u_(s)u_(s)a_(s)c_(s)c_(s)c_(s)cB 124 25655 HBV 3′ Enl neg Oallyl P = S B g_(s)g_(s)g_(s)u_(s)a_(s)a_(s)a_(s)g_(s)g_(s)u_(s)u_(s)c_(s)a_(s)u_(s)g_(s)u_(s)aB 125 25656 HBV 3′ Enl pos sc Oallyl P = S B a_(s)c_(s)c_(s)u_(s)a_(s)u_(s)c_(s)g_(s)c_(s)c_(s)u_(s)a_(s)c_(s)u_(s)c_(s)u_(s)a_(s)aB 126 25657 HBV 5′ Enl neg sc Oallyl P = S B u_(s)g_(s)a_(s)u_(s)a_(s)g_(s)c_(s)g_(s)g_(s)a_(s)u_(s)g_(s)a_(s)g_(s)a_(s)u_(s)uB 127 25658 HBV DR1 pos Oallyl P = S B u_(s)u_(s)c_(s)a_(s)c_(s)c_(s)u_(s)c_(s)u_(s)g_(s)c B 128

[0240] TABLE III A. 2.5 μmol Synthesis Cycle ABI 394 Instrument WaitTime* 2′-O- Reagent Equivalents Amount Wait Time* DNA methyl WaitTime*RNA Phosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-EthylTetrazole 23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5 sec 5 sec N-Methyl 186 233 μL  5 sec 5 sec 5 sec Imidazole TCA176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 secBeaucage 12.9 645 μL 100 sec  300 sec 300 sec Acetonitrile NA 6.67 mL NANA NA B. 0.2 μmol Synthesis Cycle ABI 394 Instrument Wait Time 2′-O-Reagent Equivalents Amount Wait Time* DNA methyl Wait Time*RNAPhosphoramidites 15 31 μL 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.731 μL 45 sec 233 min 465 sec Acetic Anhydride 655 124 μL  5 sec 5 sec  5sec N-Methyl 1245 124 μL  5 sec 5 sec  5 sec Imidazole TCA 700 732 μL 10sec 10 sec  10 sec Iodine 20.6 244 μL 15 sec 15 sec  15 sec Beaucage 7.7232 μL 100 sec  300 sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2μmol Synthesis Cycle 96 well Instrument Equivalents:DNA/ Amount:DNA/2′-O- Wait Time* 2′-O- Reagent 2′-O-methyl/Ribo methyl/Ribo WaitTime* DNA methyl Wait Time* Ribo Phosphoramidites 22/33/66 40/60/120 μL60 sec 180 sec 360 sec  S-Ethyl Tetrazole 70/105/210 40/60/120 μL 60 sec180 min 360 sec  Acetic Anhydride 265/265/265 50/50/50 μL 10 sec  10 sec10 sec N-Methyl 502/502/502 50/50/50 μL 10 sec  10 sec 10 sec ImidazoleTCA 238/475/475 250/500/500 μL 15 sec  15 sec 15 sec Iodine 6.8/6.8/6.880/80/80 μL 30 sec  30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec  Acetonitrile NA 1150/1150/1150 μL NA NA NA

[0241]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 128 <210> SEQ ID NO 1<211> LENGTH: 4 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 1 auuc 4 <210> SEQ IDNO 2 <211> LENGTH: 4 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 2 cauu 4 <210> SEQ IDNO 3 <211> LENGTH: 4 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 3 ucau 4 <210> SEQ IDNO 4 <211> LENGTH: 4 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 4 uuca 4 <210> SEQ IDNO 5 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 5 auuca 5 <210> SEQ IDNO 6 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 6 cauuc 5 <210> SEQ IDNO 7 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 7 ucauu 5 <210> SEQ IDNO 8 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 8 uucau 5 <210> SEQ IDNO 9 <211> LENGTH: 6 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 9 auucau 6 <210> SEQ IDNO 10 <211> LENGTH: 6 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 10 cauuca 6<210> SEQ ID NO 11 <211> LENGTH: 6 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 11 ucauuc6 <210> SEQ ID NO 12 <211> LENGTH: 6 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 12 uucauu6 <210> SEQ ID NO 13 <211> LENGTH: 7 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 13auucauu 7 <210> SEQ ID NO 14 <211> LENGTH: 7 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HBV Decoy Nucleic Acid <400>SEQUENCE: 14 cauucau 7 <210> SEQ ID NO 15 <211> LENGTH: 7 <212> TYPE:RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 15 ucauuca 7 <210> SEQ ID NO 16 <211> LENGTH: 7 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 16 uucauuc 7 <210> SEQ ID NO 17 <211> LENGTH: 8 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 17 auucauuc 8 <210> SEQ ID NO 18 <211> LENGTH: 8 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 18 cauucauu 8 <210> SEQ ID NO 19 <211> LENGTH: 8 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 19 ucauucau 8 <210> SEQ ID NO 20 <211> LENGTH: 8 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 20 uucauuca 8 <210> SEQ ID NO 21 <211> LENGTH: 9 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 21 auucauuca 9 <210> SEQ ID NO 22 <211> LENGTH: 9 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 22 cauucauuc 9 <210> SEQ ID NO 23 <211> LENGTH: 9 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 23 ucauucauu 9 <210> SEQ ID NO 24 <211> LENGTH: 9 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 24 uucauucau 9 <210> SEQ ID NO 25 <211> LENGTH: 10 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 25 auucauucau 10 <210> SEQ ID NO 26 <211> LENGTH: 10<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: HBV Decoy NucleicAcid <400> SEQUENCE: 26 cauucauuca 10 <210> SEQ ID NO 27 <211> LENGTH:10 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: HBV DecoyNucleic Acid <400> SEQUENCE: 27 ucauucauuc 10 <210> SEQ ID NO 28 <211>LENGTH: 10 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 28 uucauucauu 10 <210> SEQ ID NO29 <211> LENGTH: 11 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 29 auucauucau u 11<210> SEQ ID NO 30 <211> LENGTH: 11 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 30cauucauuca u 11 <210> SEQ ID NO 31 <211> LENGTH: 11 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 31 ucauucauuc a 11 <210> SEQ ID NO 32 <211> LENGTH: 11<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: HBV Decoy NucleicAcid <400> SEQUENCE: 32 uucauucauu c 11 <210> SEQ ID NO 33 <211> LENGTH:12 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: HBV DecoyNucleic Acid <400> SEQUENCE: 33 auucauucau uc 12 <210> SEQ ID NO 34<211> LENGTH: 12 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 34 cauucauuca uu 12<210> SEQ ID NO 35 <211> LENGTH: 12 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 35ucauucauuc au 12 <210> SEQ ID NO 36 <211> LENGTH: 12 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 36 uucauucauu ca 12 <210> SEQ ID NO 37 <211> LENGTH: 13<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: HBV Decoy NucleicAcid <400> SEQUENCE: 37 auucauucau uca 13 <210> SEQ ID NO 38 <211>LENGTH: 13 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 38 cauucauuca uuc 13 <210> SEQ IDNO 39 <211> LENGTH: 13 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 39ucauucauuc auu 13 <210> SEQ ID NO 40 <211> LENGTH: 13 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 40 uucauucauu cau 13 <210> SEQ ID NO 41 <211> LENGTH: 14<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: HBV Decoy NucleicAcid <400> SEQUENCE: 41 auucauucau ucau 14 <210> SEQ ID NO 42 <211>LENGTH: 14 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 42 cauucauuca uuca 14 <210> SEQID NO 43 <211> LENGTH: 14 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 43ucauucauuc auuc 14 <210> SEQ ID NO 44 <211> LENGTH: 14 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 44 uucauucauu cauu 14 <210> SEQ ID NO 45 <211> LENGTH:15 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: HBV DecoyNucleic Acid <400> SEQUENCE: 45 auucauucau ucauu 15 <210> SEQ ID NO 46<211> LENGTH: 15 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 46 cauucauuca uucau 15<210> SEQ ID NO 47 <211> LENGTH: 15 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 47ucauucauuc auuca 15 <210> SEQ ID NO 48 <211> LENGTH: 15 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 48 uucauucauu cauuc 15 <210> SEQ ID NO 49 <211> LENGTH:16 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: HBV DecoyNucleic Acid <400> SEQUENCE: 49 auucauucau ucauuc 16 <210> SEQ ID NO 50<211> LENGTH: 16 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 50 cauucauuca uucauu 16<210> SEQ ID NO 51 <211> LENGTH: 16 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 51ucauucauuc auucau 16 <210> SEQ ID NO 52 <211> LENGTH: 16 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 52 uucauucauu cauuca 16 <210> SEQ ID NO 53 <211> LENGTH:17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: HBV DecoyNucleic Acid <400> SEQUENCE: 53 auucauucau ucauuca 17 <210> SEQ ID NO 54<211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 54 cauucauuca uucauuc17 <210> SEQ ID NO 55 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 55ucauucauuc auucauu 17 <210> SEQ ID NO 56 <211> LENGTH: 17 <212> TYPE:RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 56 uucauucauu cauucau 17 <210> SEQ ID NO 57 <211>LENGTH: 18 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 57 auucauucau ucauucau 18 <210>SEQ ID NO 58 <211> LENGTH: 18 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 58cauucauuca uucauuca 18 <210> SEQ ID NO 59 <211> LENGTH: 18 <212> TYPE:RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 59 ucauucauuc auucauuc 18 <210> SEQ ID NO 60 <211>LENGTH: 18 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 60 uucauucauu cauucauu 18 <210>SEQ ID NO 61 <211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 61auucauucau ucauucauu 19 <210> SEQ ID NO 62 <211> LENGTH: 19 <212> TYPE:RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 62 cauucauuca uucauucau 19 <210> SEQ ID NO 63 <211>LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 63 ucauucauuc auucauuca 19 <210>SEQ ID NO 64 <211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 64uucauucauu cauucauuc 19 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE:RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 65 auucauucau ucauucauuc 20 <210> SEQ ID NO 66 <211>LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 66 cauucauuca uucauucauu 20 <210>SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 67ucauucauuc auucauucau 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE:RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 68 uucauucauu cauucauuca 20 <210> SEQ ID NO 69 <211>LENGTH: 21 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 69 auucauucau ucauucauuc a 21<210> SEQ ID NO 70 <211> LENGTH: 21 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 70cauucauuca uucauucauu c 21 <210> SEQ ID NO 71 <211> LENGTH: 21 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 71 ucauucauuc auucauucau u 21 <210> SEQ ID NO 72 <211>LENGTH: 21 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 72 uucauucauu cauucauuca u 21<210> SEQ ID NO 73 <211> LENGTH: 22 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 73cauucauuca uucauucauu ca 22 <210> SEQ ID NO 74 <211> LENGTH: 22 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 74 ucauucauuc auucauucau uc 22 <210> SEQ ID NO 75 <211>LENGTH: 22 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 75 uucauucauu cauucauuca uu 22<210> SEQ ID NO 76 <211> LENGTH: 23 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <400> SEQUENCE: 76ucauucauuc auucauucau uca 23 <210> SEQ ID NO 77 <211> LENGTH: 23 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<400> SEQUENCE: 77 uucauucauu cauucauuca uuc 23 <210> SEQ ID NO 78 <211>LENGTH: 24 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <400> SEQUENCE: 78 uucauucauu cauucauuca uuca 24<210> SEQ ID NO 79 <211> LENGTH: 12 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(11) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(11) <223> OTHERINFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 79 gcagagguga an 12<210> SEQ ID NO 80 <211> LENGTH: 12 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(11) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(11) <223> OTHERINFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 80 aaguggagac gn 12<210> SEQ ID NO 81 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223> OTHER INFORMATION:Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223> OTHER INFORMATION:2′-O-Allyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(5)..(5) <223> OTHER INFORMATION: n stands for inverted deoxyabasicderivative <400> SEQUENCE: 81 uucan 5 <210> SEQ ID NO 82 <211> LENGTH: 5<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: HBV Decoy NucleicAcid <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(4) <223> OTHER INFORMATION: Phosphorothioate 3′-InternucleotideLinkage <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(4) <223> OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223> OTHER INFORMATION:n stands for inverted deoxyabasic derivative <400> SEQUENCE: 82 acuun 5<210> SEQ ID NO 83 <211> LENGTH: 9 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(8) <223> OTHER INFORMATION:Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(8) <223> OTHER INFORMATION:2′-O-Allyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(9)..(9) <223> OTHER INFORMATION: n stands for inverted deoxyabasicderivative <400> SEQUENCE: 83 uucauucan 9 <210> SEQ ID NO 84 <211>LENGTH: 9 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(8) <223> OTHER INFORMATION: Phosphorothioate3′-Internucleotide Linkage <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)..(8) <223> OTHER INFORMATION: 2′-O-Allyl <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223>OTHER INFORMATION: n stands for inverted deoxyabasic derivative <400>SEQUENCE: 84 acuuacuun 9 <210> SEQ ID NO 85 <211> LENGTH: 13 <212> TYPE:RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(12)<223> OTHER INFORMATION: Phosphorothioate 3′-Internucleotide Linkage<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(12)<223> OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (13)..(13) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 85 uucauucauucan 13 <210> SEQ ID NO 86 <211> LENGTH: 13 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HBV Decoy Nucleic Acid <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(12) <223>OTHER INFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(12) <223>OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (13)..(13) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 86 acuuacuuacuun 13 <210> SEQ ID NO 87 <211> LENGTH: 17 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HBV Decoy Nucleic Acid <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(16) <223>OTHER INFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(16) <223>OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (17)..(17) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 87 uucauucauucauucan 17 <210> SEQ ID NO 88 <211> LENGTH: 17 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HBV Decoy Nucleic Acid <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(16) <223>OTHER INFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(16) <223>OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (17)..(17) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 88 acuuacuuacuuacuun 17 <210> SEQ ID NO 89 <211> LENGTH: 5 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HBV Decoy Nucleic Acid <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223>OTHER INFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223>OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: n standsfor inverted deoxyabasic derivative <400> SEQUENCE: 89 ugaan 5 <210> SEQID NO 90 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223> OTHER INFORMATION:Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223> OTHER INFORMATION:2′-O-Allyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(5)..(5) <223> OTHER INFORMATION: n stands for inverted deoxyabasicderivative <400> SEQUENCE: 90 aagun 5 <210> SEQ ID NO 91 <211> LENGTH: 8<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: HBV Decoy NucleicAcid <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(7) <223> OTHER INFORMATION: Phosphorothioate 3′-InternucleotideLinkage <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(7) <223> OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (8)..(8) <223> OTHER INFORMATION:n stands for inverted deoxyabasic derivative <400> SEQUENCE: 91 gcuugaan8 <210> SEQ ID NO 92 <211> LENGTH: 8 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(7) <223> OTHER INFORMATION:Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(7) <223> OTHER INFORMATION:2′-O-Allyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(8)..(8) <223> OTHER INFORMATION: n stands for inverted deoxyabasicderivative <400> SEQUENCE: 92 aaguucgn 8 <210> SEQ ID NO 93 <211>LENGTH: 12 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(10) <223> OTHER INFORMATION: Phosphorothioate3′-Internucleotide Linkage <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)..(11) <223> OTHER INFORMATION: 2′-O-Allyl <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (12)..(12) <223>OTHER INFORMATION: n stands for inverted deoxyabasic derivative <400>SEQUENCE: 93 ggaggcuuga an 12 <210> SEQ ID NO 94 <211> LENGTH: 12 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(11)<223> OTHER INFORMATION: Phosphorothioate 3′-Internucleotide Linkage<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(11)<223> OTHER INFORMATION: 2′-O-Allyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (12)..(12) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 94 aaguucggaggn 12 <210> SEQ ID NO 95 <211> LENGTH: 37 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Enzymatic Nucleic Acid <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223>OTHER INFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(8) <223>OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (12)..(12) <223> OTHER INFORMATION:2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(14)..(26) <223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (28)..(29) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (31)..(36) <223> OTHER INFORMATION: 2′-O-Methyl <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223>OTHER INFORMATION: 2′-deoxy-2′-C-Allyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (37)..(37) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 95 gaaaauucugaugaggccgu uaggccgaaa gagaagn 37 <210> SEQ ID NO 96 <211> LENGTH: 37<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Enzymatic NucleicAcid <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(4) <223> OTHER INFORMATION: Phosphorothioate 3′-InternucleotideLinkage <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(8) <223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (12)..(12) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (14)..(26) <223> OTHER INFORMATION: 2′-O-Methyl <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (28)..(29) <223>OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (31)..(36) <223> OTHER INFORMATION:2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(9)..(9) <223> OTHER INFORMATION: 2′-deoxy-2′-C-Allyl <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (37)..(37) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <400>SEQUENCE: 96 aaugaggcua gugacgccgu uaggcggaaa aaugaan 37 <210> SEQ ID NO97 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HBV Decoy Nucleic Acid <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)..(4) <223> OTHER INFORMATION:2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(5)..(5) <223> OTHER INFORMATION: n stands for inverted deoxyabasicderivative <400> SEQUENCE: 97 ugaan 5 <210> SEQ ID NO 98 <211> LENGTH: 5<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: HBV Decoy NucleicAcid <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(4) <223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223> OTHER INFORMATION:n stands for inverted deoxyabasic derivative <400> SEQUENCE: 98 aagun 5<210> SEQ ID NO 99 <211> LENGTH: 12 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(11) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 99 ggaggcuuga an 12<210> SEQ ID NO 100 <211> LENGTH: 12 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(11) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 100 aaguucggag gn 12<210> SEQ ID NO 101 <211> LENGTH: 8 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(7) <223> OTHER INFORMATION:2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(8)..(8) <223> OTHER INFORMATION: n stands for inverted deoxyabasicderivative <400> SEQUENCE: 101 gcuugaan 8 <210> SEQ ID NO 102 <211>LENGTH: 8 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:HBV Decoy Nucleic Acid <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(7) <223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (8)..(8) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <400>SEQUENCE: 102 aaguucgn 8 <210> SEQ ID NO 103 <211> LENGTH: 12 <212>TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(11)<223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (12)..(12) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 103gcagagguga an 12 <210> SEQ ID NO 104 <211> LENGTH: 12 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(11)<223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (12)..(12) <223> OTHER INFORMATION: nstands for inverted deoxyabasic derivative <400> SEQUENCE: 104aaguggagac gn 12 <210> SEQ ID NO 105 <211> LENGTH: 5 <212> TYPE: RNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: HBV Decoy Nucleic Acid<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(4)<223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: n standsfor inverted deoxyabasic derivative <400> SEQUENCE: 105 uucan 5 <210>SEQ ID NO 106 <211> LENGTH: 5 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223> OTHER INFORMATION:2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(5)..(5) <223> OTHER INFORMATION: n stands for inverted deoxyabasicderivative <400> SEQUENCE: 106 acuun 5 <210> SEQ ID NO 107 <211> LENGTH:9 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: HBV DecoyNucleic Acid <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(8) <223> OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION:n stands for inverted deoxyabasic derivative <400> SEQUENCE: 107uucauucan 9 <210> SEQ ID NO 108 <211> LENGTH: 9 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HBV Decoy Nucleic Acid <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(8) <223>OTHER INFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n standsfor inverted deoxyabasic derivative <400> SEQUENCE: 108 acuuacuun 9<210> SEQ ID NO 109 <211> LENGTH: 13 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(12) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (13)..(13) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 109 uucauucauu can 13<210> SEQ ID NO 110 <211> LENGTH: 13 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(12) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (13)..(13) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 110 acuuacuuac uun 13<210> SEQ ID NO 111 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(16) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (17)..(17) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 111 uucauucauu cauucan17 <210> SEQ ID NO 112 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV Decoy Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(16) <223> OTHERINFORMATION: 2′-O-Methyl <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (17)..(17) <223> OTHER INFORMATION: n stands forinverted deoxyabasic derivative <400> SEQUENCE: 112 acuuacuuac uuacuun17 <210> SEQ ID NO 113 <211> LENGTH: 18 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(18) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(16) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 113 nuuucuaagu aaacagun 18<210> SEQ ID NO 114 <211> LENGTH: 18 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(18) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(16) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 114 nacuguuuac uuagaaan 18<210> SEQ ID NO 115 <211> LENGTH: 18 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(18) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(16) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 115 naaguaacuc uauguuan 18<210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(20) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(19) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 116 nuacaugaac cuuuaccccn 20<210> SEQ ID NO 117 <211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 117 nggguaaagg uucauguan 19<210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(20) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(19) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 118 naccuaucgc cuacucuaan 20<210> SEQ ID NO 119 <211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 119 nugauagcgg augagauun 19<210> SEQ ID NO 120 <211> LENGTH: 13 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(13) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(11) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(12) <223> OTHERINFORMATION: 2′-O-Methyl <400> SEQUENCE: 120 nuucaccucu gcn 13 <210> SEQID NO 121 <211> LENGTH: 18 <212> TYPE: RNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHER INFORMATION:n stands for inverted deoxyabasic derivative <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (18)..(18) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(16) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 121 nuuucuaagu aaacagun 18 <210>SEQ ID NO 122 <211> LENGTH: 18 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(18) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(16) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 122 nacuguuuac uuagaaan 18 <210>SEQ ID NO 123 <211> LENGTH: 18 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(18) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(16) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 123 naaguaacuc uauguuan 18 <210>SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(20) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(19) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 124 nuacaugaac cuuuaccccn 20<210> SEQ ID NO 125 <211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 125 nggguaaagg uucauguan 19<210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (20)..(20) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(19) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 126 naccuaucgc cuacucuaan 20<210> SEQ ID NO 127 <211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(17) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(18) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 127 nugauagcgg augagauun 19<210> SEQ ID NO 128 <211> LENGTH: 13 <212> TYPE: RNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HBV DNA Binding Nucleic Acid <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(13) <223> OTHERINFORMATION: n stands for inverted deoxyabasic derivative <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(11) <223> OTHERINFORMATION: Phosphorothioate 3′-Internucleotide Linkage <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(12) <223> OTHERINFORMATION: 2′-O-Allyl <400> SEQUENCE: 128 nuucaccucu gcn 13

What we claim is:
 1. A nucleic acid molecule that specifically binds thehepatitis B virus (HBV) reverse transcriptase primer, wherein saidnucleic acid molecule comprises the sequence (UUCA)n, wherein n is aninteger from 1 to
 10. 2. A nucleic acid molecule that specifically bindsthe hepatitis B virus (HBV) reverse transcriptase primer, wherein saidnucleic acid molecule comprises any of SEQ ID NOs: 1-79, 81, 83, 85, 87,89, 91, 93, 97, 99, 101, 103, 105, 107, 109, and
 111. 3. A nucleic acidmolecule that specifically binds to the Enhancer I sequence of HBV DNA.4. A nucleic acid molecule that specifically binds to the Enhancer Iregion of HBV DNA, wherein said nucleic acid molecule comprises any ofSEQ ID NOs: 113, 116, 118, 120, 121, 124, 126, and
 128. 5. A method ofadministering to a cell a nucleic molecule of any of claims 1-4comprising contacting said cell with the nucleic acid molecule underconditions suitable for said administration.
 6. The method of claim 5,further comprising administering one or more other therapeutic compoundsunder conditions suitable for said administration.
 7. The method ofclaim 6, wherein said other therapeutic compound is type I interferon.8. The method of claim 6, wherein said other therapeutic compound is3TC® (Lamivudine).
 9. The method of claim 6, wherein said othertherapeutic compound and the nucleic acid molecule are administeredsimultaneously.
 10. The method of claim 6, wherein said othertherapeutic compound and nucleic acid molecule are administeredseparately.
 11. The method of claim 7, wherein said type I interferon ischosen from interferon alpha, interferon beta, consensus interferon,polyethylene glycol interferon, polyethylene glycol interferon alpha 2a,polyethylene glycol interferon alpha 2b, and polyethylene glycolconsensus interferon
 12. The nucleic acid molecule of any of claims 1-4,wherein said nucleic acid molecule comprises a nucleic acid backbonemodification.
 13. The nucleic acid decoy molecule of any of claims 1-4,wherein said nucleic acid molecule comprises a nucleic acid sugarmodification.
 14. The nucleic acid decoy molecule of any of claims 1-4,wherein said nucleic acid molecule comprises a nucleic acid basemodification.
 15. The method of claim 5, wherein said cell is amammalian cell.
 16. The method of claim 6, wherein said cell is amammalian cell.
 17. The method of claim 15, wherein said cell is a humancell.
 18. The method of claim 16, wherein said cell is a human cell. 19.The method of claim 15, wherein said administration is in the presenceof a delivery reagent.
 20. The method of claim 16, wherein saidadministration is in the presence of a delivery reagent.
 21. The methodof claim 19, wherein said delivery reagent is a lipid.
 22. The method ofclaim 21, wherein said lipid is a cationic lipid or a phospholipid. 23.The method of claim 19, wherein said delivery reagent is a liposome. 24.The method of claim 20, wherein said delivery reagent is a lipid. 25.The method of claim 24, wherein said lipid is a cationic lipid or aphospholipid.
 26. The method of claim 20, wherein said delivery reagentis a lipsome.
 27. The nucleic acid molecule of claim 1, wherein saidnucleic acid molecule is a decoy nucleic acid molecule.
 28. The nucleicacid molecule of claim 1, wherein said nucleic acid molecule is anaptamer nucleic acid molecule.
 29. The nucleic acid molecule of claim 3,wherein said Enhancer I sequence comprises a Hepatocyte Nuclear Factor 3and/or Hepatocyte Nuclear Factor 4 binding sequence.